Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom

ABSTRACT

The present invention is directed to novel nucleotide and amino acid sequences of Porcine Epidemic Diarrhea Virus (“PEDV”), including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals. Vaccines provided according to the practice of the invention are effective against multiple swine PEDV genotypes and isolates. Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines. Particularly important aspects of the invention include polynucleotide constructs that replicate in tissue culture and in host swine. The invention also provides for novel full length PEDV genomes that can replicate efficiently in host animals and tissue culture.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is a national stage of PCT applicationPCT/US2016/019846 filed on Feb. 26, 2016, which in turn claims priorityunder 35 U.S.C § 119 to Provisional Patent Application Ser. No.62/121,955 filed Feb. 27, 2015, Provisional Patent Application Ser. No.62/209,119 filed Aug. 24, 2015, Provisional Patent Application Ser. No.62/250,961 filed Nov. 4, 2015 and Provisional Patent Application Ser.No. 62/276,022 filed Jan. 7, 2016 herein incorporated by reference intheir entirety.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

The claimed invention was made as a result of activities undertakenwithin the scope of a joint research agreement between Zoetis ServicesLLC and Iowa State University Research Foundation.

FIELD OF THE INVENTION

The present invention is directed to novel immunogenic compositions thatprotect swine from disease caused by porcine epidemic diarrhea virus(PEDV).

BACKGROUND OF THE INVENTION

Porcine epidemic diarrhea (PED) is highly contagious and ischaracterized by dehydration, diarrhea, and high mortality in swine,particularly young piglets. The causative agent, porcine epidemicdiarrhea virus (PEDV), is a single stranded, positive sense RNA virusbelonging to the Alphacoronavirus genus of the family Coronaviridae.PEDV has a total genome size of approximately 28 kb and contains 7 openreading frames. Symptoms of PEDV infection are often similar to thosecaused by transmissible gastroenteritis virus (TGEV) and porcinedeltacoronavirus (PDCoV), both of which are also members of theCoronaviridae, It should be noted that cross protection between PEDV andTGEV is not generally observed, the overall viral nucleotide sequencesbeing at most about 60% similar.

PED was likely first observed in Europe circa 1970, and the causativevirus was subsequently characterized (see for example M. Pensaert et al.Arch. Virol, v. 58, pp 243-247, 1978 and D. Chasey et al., Res. Vet Sci,v. 25, pp 255-256, 1978). PEDV was not identified in North America until2013, at which point widespread outbreaks commenced, and severe economiclosses to the swine industry resulted. The virus appeared in multiple,widely distributed sow herds within days, and it has spread to at least32 states. Producers can expect losses of up to 100% in naïve neonatalpiglets. Present recommendations for management of infection includeimplementation of strict biosecurity and/or intentional exposure of thewhole herd to PEDV to accomplish immunity.

PEDV caused widespread epidemics in several European countries duringthe 1970s and 1980s; but since the 1990s PED has become rare in Europewith occasional outbreak. This classical PEDV strain subsequently wasspread to Asian countries such as Japan, China, South Korea, etc. Since2010, severe PED epizootic outbreaks have been reported in China and thePEDV recovered from these outbreaks were genetically different from theclassical PEDV strains. The initial PED outbreaks in U.S. swine hadsimilar clinical presentations to those observed in China. Sequenceanalyses revealed that the original U.S. PEDVs (hereafter designated asU.S. PEDV prototype strain) are most genetically similar to some PEDVscirculating in China in 2011-2102. In January 2014, a PEDV variantstrain, which has insertions and deletions (INDEL) in the spike genecompared to the U.S. PEDV prototype strains, was identified in the U.S.swine population. This variant strain was designated as U.S. PEDVS-INDEL-variant strain. After the PED outbreak in the U.S., detection ofU.S. prototype-like PEDV has been reported in Canada, Mexico, Taiwan,South Korea, and Japan; detection of U.S. S-INDEL-variant-like PEDV hasbeen reported in South Korea, Japan, Germany, Belgium, France, andPortugal. Currently, PEDV remains as a significant threat to the globalswine industry.

PEDV generally grows poorly in culture, and there is a need to identifyboth particular strains that are appropriate for the culturing ofsufficient virus for commercial vaccine preparation. Additionally, thereis a need to develop vaccines that provide effective cross protectionagainst known isolates of PEDV, and which are expected to provideeffective cross protection against evolving PEDV strains worldwide.

SUMMARY OF THE INVENTION

The present invention encompasses immunogenic compositions comprisingvariant PEDV strains passaged from prototype strain and from indelstrains. The U.S. PEDV S-INDEL-variant strains are genetically differentfrom the U.S. PEDV prototype-like strains, including characteristicinsertions and deletions in the spike protein S1 domain region,particularly the first 1170 bases thereof. The insertions and deletionsinclude 3 deletions (a 1-nt deletion at position 167, an 11-nt deletionat position 176, and a 3-nt deletion at position 416), a 6-nt insertionbetween positions 474 and 475, and several other mutations mainlylocated in the first 1,170 nucleotides of the S1 region. The U.S. PEDVS-INDEL-variant strains are less virulent than the U.S. PEDV prototypestrains and may be used, in one embodiment or whole virus, as attenuatedlive vaccines. Pigs infected with live attenuated serially passagedS-INDEL-variant strains of the invention do not cause disease whenadministered to piglets and, importantly, exhibited cross protectionagainst challenge with either the virulent prototype PEDV strains or theS-INDEL-variant strains.

Thus, the invention comprises an immunogenic composition, suitable to beused as a vaccine, which comprises a S-INDEL-variant PEDV strain of theinvention, preferably live and attenuated, or an immunogenic fragmentthereof, one or more adjuvants, and optionally one or more excipients,in an amount effective to elicit production of neutralizing antibodiesin swine. The adjuvant preferably provides an oil-in-water emulsion withadditional components. The immunogenic compositions of the inventionprotect swine from infection by PEDV, and are effective in single doses,in two-dose programs, or in vaccination programs involving multipledoses, which may be spread apart by at least a week, and optionally atgreater intervals of time, such as one to several months. It should benoted that depending on the level of epidemic threat in a particularswine population, the vaccine dose program of one, two, or multipledoses may be repeated, from time to time, as a precautionary measure.Additionally, it should be noted that vaccinating a mother sow duringpregnancy will provide protection to a young piglet, via maternaltransfer of antibodies and T-cells in colostrum and milk, although suchprotection may need to be followed up with additional vaccination dosesto the piglet. Vaccination of all swine, including piglets and adults iscontemplated.

It has surprisingly been found that the U.S. PEDV S-INDEL-variant strainof the invention provides cross reactivity/protection against other PEDVstrains and it is expected that protection will be conferred againstEuropean strains generally, and more particularly against the recentlyemerging isolates in Europe that had very high genetic similarity (>99%)to the U.S. S-INDEL-variant strains at the whole genome sequence level.Accordingly, the vaccinating compositions of the present invention areuseful to protect swine from disease or challenge by European strains ofPEDV generally, including recent isolates.

The present invention includes novel nucleotide and amino acid sequencesof PEDV, including novel genotypes thereof, all of which are useful inthe preparation of vaccines for treating and preventing diseases inswine and other animals. Vaccines provided according to the practice ofthe invention are effective against multiple swine PEDV genotypes andisolates. Diagnostic and therapeutic polyclonal and monoclonalantibodies are also a feature of the present invention, as areinfectious clones useful in the propagation of the virus and in thepreparation of vaccines. Of particular importance, there are disclosedvaccines that comprise, as antigen, a whole virus (live or attenuated)or an single antigenic protein of a PEDV open reading frame, mostparticularly from the first 2.2 kb of the spike gene, more particularlythe S1 domain, and also fragments of the full length sequence encodingthe PEDV proteins. The invention also provides the full length genomicsequences of PEDV strains at different passages in cell culture that canreplicate efficiently in host animals and tissue culture.

The present invention provides a method of treating or preventing adisease or disorder in an animal caused by infection with PEDV,including disease states that are directly caused by PEDV, and diseasestates contributed to or potentiated by PEDV. Disease states in swinethat may be potentiated by PEDV, and which may also be treated orprevented according to the practice of the invention, include thosecaused by or associated with Transmissible Gastroenteritis virus, PHEV,and PRCV.

The present invention also includes the option to administer acombination vaccine, that is, a bivalent or multivalent combination ofantigens, which may include live, modified live, or inactivated antigensagainst the non-PEDV pathogen, with appropriate choice of adjuvant.

Based in part upon the unique PEDV sequences as disclosed herein, thepresent invention also provides a diagnostic kit for differentiatingbetween porcine animals vaccinated with the above described PEDVvaccines and porcine animals infected with field strains of PEDV.

Representative embodiments of the invention include an isolatedpolynucleotide sequence that includes a genomic polynucleotide whichencodes variant PEDV proteins which are attenuated and may be used as animmunogenic composition. This can include whole genome sequencesselected from the group consisting of:

(a) SEQ ID NOS 8-16, and 35-39 or an immunogenic fragment thereof thatencodes the PEDV virus variants;

(b) the complement of any sequence in (a);

(c) a polynucleotide that hybridizes with a sequence of (a) or (b) understringent conditions defined as hybridizing to filter bound DNA in 0.5MNaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C.

(d) a polynucleotide that is at least 70% identical to thepolynucleotide of (a) or (b);

(e) a polynucleotide that is at least 80% identical to thepolynucleotide of (a) or (b);

(f) a polynucleotide that is at least 90% identical to thepolynucleotide of (a) or (b); and

(g) a polynucleotide that is at least 95% identical to thepolynucleotide of (a) or (b)

Preferably in combination with a second heterologous sequence.

The invention further provides RNA and DNA molecules, their complements,fragments and vectors and plasmids for the expression of any such RNA orDNA polynucleotides, and for PEDV virus that is expressed from suchnucleotide sequences, wherein said virus is live, or fully or partiallyattenuated.

The invention also provides a vaccine that comprises a polynucleotidesequence as aforementioned, and corresponding nucleotide sequences thatmay function as infectious clones.

The invention also includes nucleic acid ORFs which encode variantproteins from PEDV as reflected in Table 1 herewith. Table 1 variationsare described with reference to PEDV USA/IL20697/2014 passage 5.

TABLE 1Nucleotide and amino acid changes of PEDV isolate IL20697/2014 during serial passages in cell cultureGenome region Nucleotide in^(a): or ORF Position P18R-1 P18R-1 P18R-1(nucleotide Encoded in whole 89G8b 94F6a 92P6a Virus position)^(a)protein genome P3 P5 P7 P18 P30 P38 P45 P60 P3R1 P5R1 P7R1 P8R1 ZoetisZoetis Zoetis PEDV 5'UTR (1-292) None USA/ ORF1a (293- pp1a;    424 C CC C C C C C T T T T T T T IL20697/2014 12646); pp1ab   781 G T T T T T TT TG G G G G G G ORF1a/1b  1944 T T T T T T T T T T T T T C C(293-12616),  2083 T T T T T T C C T T T T T T T (12616-20637)  3610 T TT T T T C C T T T T T T T  4400 T C C C C C C C C C C C C C C  5064 A AA A A T T T A A A A A A A  6367 T T T T T C C C T T T T T T T  7435 A AG G G G G G A A A A A A A 11080 C C C C C T T T C C C C C C C 12391 C CC C C C C C C C C C T T T 13387 C T T T T T T T C C C C C C C 13992 T TT T T T T T G G G G G G G 14691 C C C C C C C C T T T T T T T 15550- GTGT GT GT GT TC TC TC GT GT GT GT GT GT GT 15551 18704 A A A A A G G G AA A A A A A S (20634- Spike 20717 C C C C T T T T C C C C C C C 24785)20775 G G G G G G G G T T T T T T T 21083 T A A A A A A A A A A A A A A21667 C C C C C C C C T T T T T T T 22232 C C C C C C C C T T T T T T T22530 G G A A G G G G G G G G G G G 22919 C C C C T T T T C C C C C C C23283 A A A C C C C C A A A A A A A 23509 T T T T C C C C T T T T T T T23550 T T T T T T T C T T T T C C C 23658 T T T T T T T T T T C C C C C24307 T T T T T A A A T T T T T T T 24448 TC T T T T T T T C C C C C C CORFS  Hypo- 24808- CACGA CACGA CACGA CACGA CACGA CACGA — — CACGA CACGACACGA CACGA CACGA CACGA CACGA (24785-2549)^(d) thetical 24823 TTGACTTGAC TTGAC TTGAC TTGAC TTGAC — — TTGAC TTGAC TTGAC TTGAC TTGAC TTGACTTGAC protein ACAGT ACAGT ACAGT ACAGT ACAGT ACAGT ACAGT ACAGT ACAGTACAGT ACAGT ACAGT ACAGT 3 T T T T T T T T T T T T T 25197- ATTAT ATTATATTAT ATTAT ATTAT AT--- AT--- AT--- ATTAT AT--- ----T ----T ----T ----T----T 25201 C C C C C T T T C C C C C C C 25304- CGTGG CGTGG CGTGG CGTGGCGTGG CGTGG CGTGG CGTGG CGTGG 25359 GCGGC GCGGC GCGGC GCGGC GCGGC GCGGCGCGGC GCGGC GCGGC AAGAA AAGAA AAGAA AAGAA AAGAA AAGAA AAGAA AAGAA AAGAAGCTGA GCTGA — — — — — — GCTGA GCTGA GCTGA GCTGA GCTGA GCTGA GCTGA CCTACCCTAC — — — — — — CCTAC CCTAC CCTAC CCTAC CCTAC CCTAC CCTAC AGCTG AGCTG— — — — — — AGCTG AGCTG AGCTG AGCTG AGCTG AGCTG AGCTG TTGCG TTGCG — — —— — — TTGCG TTGCG TTGCG TTGCG TTGCG TTGCG TTGCG AACTG AACTG — — — — — —AACTG AACTG AACTG AACTG AACTG AACTG AACTG TTGAG TTGAG TTGAG TTGAG TTGAGTTGAG TTGAG TTGAG TTGAG CTTCTT CTTCTT CTTCTT CTTCTT CTTCTT CTTCTT CTTCTTCTTCTT CTTCTT GATGG GATGG GATGG GATGG GATGG GATGG GATGG GATGG GATGGE(25440-25670) Envelope 25624 C C C C C C T T C C C C C C CM(25678-26358) Membrane 25690 T T T T T T T G T T T T T T T 26224 G G GG G G A A G G G G G G G N(26370-27695) Nucleo- 26448 G G G G G G G G C CC C C C C capsid 26864 C T T T T C T T C C C C C C C 3′UTR(26370- None27695) ^(a)Nucleotides are numbered according to the PEDVUSA/IL20697/2014 P3 sequences. ^(b)Only nonsynonymous mutations areshown and sile mutations are not shown. Amino acids of replicaseproteins are numbered according to their locations in the replicasepolyprotein pp1ab. ^(c), stop codon. ^(d)Compared to the P3, P5, P18 andP30 viruses as well as PrR1, P5R1, P8R1 viruses, the P45 and P60 viruseshad 16-nucleotide deletion at the nucleotide positions 24,803-24,823,resulting in early stop of ORF3 translation of the P45 and P60 viruses.Basically the deduced P45 and P60 ORF3 proteins are only 7 amino acidlong. Compared to the P5 virus, the P7, P8, P18 viruses had 4-nucleotidedeleta (ATTA) at the nucleotide positions 25,197-25,200, resulting inearly stop of ORF3 translations of the P7, P8, and P18 viruses.Basically the deduced P7, P8 and P18 ORF3 proteins are 143 aa long.Compared to the P3, P5, P7, P18 and P30 virues, the P45 and P60 viruseshad 3-nucelotide deletion (TAT) at the nucleotide positions25,199-25,201. Compared to the P3R1 virus, the P5R1 virus had3-nucleotide deletion (TAT) at the nucleotide postions 25,199-25,201.Compared to the P3R1 virus, the P5R1, P7R1, and P18R1 viruses had4-nucleotide deletion (ATTA) at the nucleotide positions 25,197-25,200.Compared to the P3 and P5 viruses as well at P3R1, P5R1, P7R1, and P18R1viruses, the P7, P18, P30, P45 and P60 virus had 56-nucleotide deletionat the nucleotide positions 25,304-25,35

The invention further provides nucleic acid sequences and resultantprotein variants that have amino acid substitutions and which reducevirulence, cause attenuation and allow the compositions to be usedsafely as immunogenic compositions and as vaccines.

Amino acid sequences and variant proteins are shown in Table 2 herewithagain, amino acid references are made with respect to passage 5 ofPEDV/USA/IL20697/2014.

In a further preferred embodiment, and taking advantage of thesubstantial polypeptide sequence information disclosed herein, there arefurther provided polypeptide vaccines wherein the antigen is defined by(a) the spike protein; or (b) an amino acid sequence that is at least 90percent identical thereto; or (c) an arginine rich region thereof.

In a further embodiment the invention includes vaccine compositionscomprising a a live attenuated variant strain porcine epidemic diarrheavirus (PEDV), and a carrier, wherein said composition is capable ofprotecting swine from challenge by both variant and prototype strains ofPEDV and preventing or treating one or more of symptoms associated withPEDV infection, and wherein achievement of protection is determined byan endpoint selected from the group consisting of prevention or controlof any of the PEDV infection symptoms of dehydration, fever, diarrhea,vomiting, poor lactational performance, poor reproduction performance,mortality, and prevention or control of weight loss or failure to gainweight, wherein said strain encodes proteins with a substitution of thefollowing amino acids and their conservative variants and proteins withthe specific substitutions and 99% homology to the remainder of thesequences: acid substitutions include the following or theirconservative variants:

A) Passage P18R1 F6a

-   -   polyprotein 1a/1b (SEQ ID NO: 46): P at position 551 (P        immediately after DEDAT);

spike protein (SEQ ID NO:47): P at position 1009 (P immediately afterIGNIT), H at position 973 (H immediately after ALPFS), L at position 48(L immediately after APAVV), V at position 345 (A immediately afterTNLSF);

ORF 3 (SEQ ID NO: 48): deletion of I at position 144 (I immediatelyafter YDGKS), deletion of 174-189 (immediately after LYLAI), LTANPL atposition 138-143 (immediately after NGKAA), and,

nucleocapsid protein (SEQ ID NO: 51) H at position 27 (H immediatelyafter LRVTN).

B) Passage P18R1 Gb8

-   -   Spike protein (SEQ ID NO:47): P at position 1009 (P immediately        after IGNIT), H at position 973 (H immediately after ALPFS), L        at position 48 (L immediately after APAVV), V at position 345(A        immediately after TNLSF);

ORF 3 (SEQ ID NO: 48): deletion of I at position 144 (I immediatelyafter YDGKS), deletion of 174-189 (immediately after LYLAI), LTANPL atposition 138-143 (immediately after NGKAA), and,

nucleocapsid protein (SEQ ID NO: 51) H at position 27 (H immediatelyafter LRVTN).

C) Passage P7

spike protein (SEQ ID NO:47): K at position 633 (K immediately afterTPKPL);

ORF 3 (SEQ ID NO:48): deletion of L at position 189 (L immediately afterTANPL).

D) Passage P18

-   -   spike protein (SEQ ID NO:47): K at position 633 (K immediately        after TPKPL), R at position 884 (R immediately after VYDPA);

ORF 3 (SEQ ID NO:48): deletion of L at position 189 (L immediately afterTANPL).

E) Passage P30

-   -   spike protein (SEQ ID NO:47): R at position 884 (R immediately        after VYDPA), A at position 959 (A immediately after LIGGM);

ORF 3 (SEQ ID NO:48): deletion of L at position 189 (L immediately afterTANPL).

F) Passage P38

-   -   spike protein (SEQ ID NO:47): L at position 48 (L immediately        after APAVV), V at position 345 (immediately after TNLSF), R at        position 884 (R immediately after VYDPA), A at position 959 (A        immediately after LIGGM),

ORF 3 (SEQ ID NO:48): deletion of 138-144(immediately after NGKAA)deletion of L at 189 (L immediately after TANPL),

Nucleocapsid (SEQ ID NO:51) H at position 27 (H immediately afterLRVTN).

G) Passage P45

polyprotein 1a/1b (SEQ ID NO:46): M at position 1591 (M immediatelyafter VVKVS), S at position 5087 (S immediately after YLFST), S atposition 6138 (S immediately after WQTFS);

spike protein (SEQ ID NO:47): R at position 884 (R immediately afterVYDPA), A at position 959 (A immediately after LIGGM), D at position1225 (D immediately after IESLV);

ORF 3 (SEQ ID NO:48): deletion of Y at position 8 (Y immediately afterLGLFO), deletion of 138-144 (immediately after NGKAA), and deletion of174 to 189 189 (immediately after LYLAI),

envelope protein (SEQ ID NO:49) F at position 62 (F immediately afterYRVYK);

membrane protein I at position 183 (I immediately after IVYGG).

H) Passage P60

polyprotein 1a/1b (SEQ ID NO:46): M at position 1591 (M immediatelyafter VVKVS), S at position 5087 (S immediately after YLFST), S atposition 6138 (S immediately after WQTFS);

spike protein (SEQ ID NO:47): R at position 884 (immediately afterVYDPA), A at position 959 (immediately after LIGGM), H at position 973(H immediately after ALPFS), D at position 1225 (D immediately afterIESLV);

ORF 3 (SEQ ID NO:48): deletion of Y at position 8 (Y immediately afterLGLFO), deletion of 138-144 (immediately after NGKAA), and deletion of174 to 189 (immediately after LYLAI),

envelope protein (SEQ ID NO:49) F at position 62 (F immediately afterYRVYK);

membrane protein A at position 5 (A immediately after MSNG), I atposition 183(I immediately after IVYGG).

I) Passage P3R1

spike protein (SEQ ID NO:47): L at position 48 (immediately afterAPAVV), V at position 345 (immediately after TNLSF); T at position 1272(T immediately after DVFNA)

envelope protein (SEQ ID NO:49) S at position 62 (S immediately afterYRVYK);

nucleocapsid protein (SEQ ID NO:51): H at position 27 (H immediatelyafter LRVTN).

The invention further includes passages and amino acid substitution frompassaging the prototype virus, deposited at Genbank® Accesssion KF650371Passage 3. Variations in amino acids are reflected the following:

polyprotein 1a/1b (SEQ ID NO:52): V at 814, A at 1076, F at 1564, I at1896, H at 2310 Y at 2600, Fat 3247, Vat 3473, or Rat 3522;

spike protein (SEQ ID NO:54): N at 257, I at 326, F at 375, Y at 491, Rat 881, R at 888 F at 1277, Tat 1339, or L at 1358;

ORF 3 (SEQ ID NO:55) stop at 39 or later;

Envelope protein (SEQ ID NO:56) position I at 69;

membrane protein (SEQ ID NO: 57) position T at 208;

nucleocapsid protein (SEQ ID NO: 58) position L at 141, Q at 418, N at424, or I at 439.

It is within the scope of the invention to make additional modificationsin any PEDV passages described herein, wherein said modificationsinclude introducing mutations of another passage (Prototype, INDELlineage 1, or INDEL lineage 2 and combinations thereof) described hereinusing known methods according to one of skill in the art, for example,homologous recombination.

GenBank® is the recognized United States-NIH genetic sequence database,comprising an annotated collection of publicly available DNA sequences,and which further incorporates submissions from the European MolecularBiology Laboratory (EMBL) and the DNA DataBank of Japan (DDBJ), seeNucleic Acids Research, January 2013, v 41(D1) D36-42 for discussion.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 shows a phylogenetic analysis of the PEDV S1 portion nucleotidesequences from clinical cases in January 2014 when our group firstidentified U.S. PEDV S-INDEL-variant strain. The tree was constructedusing the distance-based neighbor-joining method of the software MEGA5.2. Bootstrap analysis was carried out on 1,000 replicate data sets,and values are indicated adjacent to the branching points. The newlyidentified U.S. PEDV S-INDEL-variant strains (ISU cases 1-5) areindicated with solid circles that had sequences different from PEDVidentified in US in 2013 (U.S. PEDV prototype strain) which areindicated with triangles.

FIG. 2 shows phylogenetic trees based on the S1 portion sequences andthe whole genome sequences. See the attachment. FIG. 2A illustratesPhylogenetic analysis of 52 global PEDVs based on the S1 portionsequences. FIG. 2B illustrates Phylogenetic analysis of 51 global PEDVsbased on the whole genome sequences. One can see that, in addition to inUS swine, the US prototype-like strains have been detected in SouthKorea, Canada and Mexico; the US variant-INDEL-like strains have beendetected in South Korea, Mexico and Germany.

FIG. 3A shows the S1 sequence of the strain of the invention (SEQ ID NO:1). FIG. 3B shows the whole genome sequence of the S-INDEL-variant PEDVcell culture isolate 2014020697-P5 passage 5, lineage 1 (SEQ ID NO:8).

FIGS. 4A-4I are an alignment comparing the spike gene sequences of thePEDV US S-INDEL-variant isolate 2014020697-P5 passage 5, lineage 1 (SEQID NO:2) to the PEDV US prototype isolates 2013019338-P3 passage 3 (SEQID NO:3), 2013022038-P3 passage 3 (SEQ ID NO:4), 2013035140-P3 passage 3(SEQ ID NO:5), 2013049379-P3 passage 3 (SEQ ID NO:6), and 2013049469-P1passage 1 (SEQ ID NO:7), identical bases are indicated with dots.

FIGS. 5A-5EEE are a comparison of the whole genome of PEDV US variantisolate 2014020697-P5 passage 5, lineage 1 (SEQ ID NO:8) to the PEDV USprototype isolates 2013019338-P3 passage 3 (SEQ ID NO:9), 2013022038-P3passage 3 (SEQ ID NO:10), 2013035140-P3 passage 3 (SEQ ID NO:11),2013049379-P3 passage 3 (SEQ ID NO:12), and 2013049469-P1 passage 1 (SEQID NO:13), identical bases are indicated with dots.

FIGS. 6A-6D are the phylogenetic analysis of the full-length genome andS1 portion nucleotide sequences. Three U.S. prototype PEDV isolatesobtained in this study (USA/NC35140/2013, USA/IA49379/2013 andUSA/NC49469/2013) and two isolates previously isolated but evaluated inthis study (U.S. PEDV prototype isolate USA/IN19338/2013 and U.S.S-INDEL-variant isolate USA/IL20697) are indicated with bullet points ortriangle. Forty-five PEDV sequences selected from GenBank® were alsoincluded for analysis. The trees were constructed using thedistance-based neighbor-joining method (6A and 6C) and the maximumlikelihood method (FIG. 6B and FIG. 6D) of the software MEGA6. The U.S.prototype-like PEDVs are shown in blue color fonts with clade 1 andclade 2 indicated. The U.S. S-INDEL-variant-like PEDVs are shown in redcolor fonts.

FIG. 7A-7D show the clinical assessment and virus shedding of 5-day-oldpigs inoculated with U.S. PEDV prototype and S-INDEL-variant isolates.FIG. 7A shows the average diarrhea scores over the course of 7 days postinoculation (DPI). FIG. 7B shows the average daily weight gain (ADG).Statistical analyses on ADG were performed among groups from (−1) to 3DPI and from (−1) to 7 DPI, respectively. Virus shedding in rectal swabsare shown in FIG. 7C and in sera in FIG. 7D of inoculated pigs wasdetermined by a quantitative PEDV N gene-based real-time RT-PCR. Thevirus titers (Log 10Genomic copies/ml) at each time point were meanvirus titers of all available pigs (both PCR-positive and negative pigs)in each group. Standard error bars are shown in each figure. In FIG. 7D,viremia levels among the groups were statistically analyzed at 3 DPI and7 DPI, respectively. Labels without the same letters indicatesignificant differences, for example A and B have significant differencebut A and AB have no significant differences.

FIG. 8A-8B show the examination of gross lesions of inoculated pigs at 3DPI and 7 DPI necropsies. Average scores of contents of small intestine,cecum and colon are shown in FIG. 8A. Average scores of lesions of smallintestine, cecum and colon are shown in FIG. 8B. The scoring criteriawere described in Materials and Methods Example 2. Statistical analyseswere performed on various inoculated groups, but each time on one tissuetype and on one time point. Labels without the same letters indicatesignificant differences.

FIG. 9A-9T show representative microscopic lesions and IHC staining ofinoculated pigs necropsied at 3 DPI. Hematoxylin and eosin-stainedtissue sections of small intestine (ileum) from different inoculationgroup (FIG. 9A-9E). Immunohistochemistry-stained tissue sections ofileum (FIG. 9F-9J), cecum (FIG. 9K-9O), and colon (FIG. 9P-9T) fromdifferent inoculation group. All images are in 100× magnification.

FIG. 10A-10D shows the mean villus height (μm), crypt depth (μm),villus/crypt ratio, and immunohistochemistry scores of pigs necropsiedat 3 DPI. Statistical analyses were performed on various inoculatedgroups, but each time on one tissue type. Labels without the sameletters indicate significant differences.

FIG. 11A-11D shows the mean villus height (μm), crypt depth (μm),villus/crypt ratio, and immunohistochemistry scores of pigs necropsiedat 7 DPI. Statistical analyses were performed on various inoculatedgroups, but each time on one tissue type. Labels without the sameletters indicate significant differences.

FIG. 12 is a schematic diagrams of PEDV genome organization and putativefunctions of viral proteins. The PEDV entire genome organization isdepicted (top). The 5′ leader, ORFs 1a and 1b encoding replicasepolyproteins, spike (S), ORF3, envelope (E), membrane (M) andnucleocapsid (N) genes are shown, with the ribosomal frameshift siteindicated. Predicted cleavage products (nsp1-nsp16) of the replicasepolyproteins and putative functional domains are depicted (bottom). Nsp1may possibly suppress the innate immune response and host proteinsynthesis. The exact function of nsp2 remains unclear. Nsp3 includespapain-like proteases (PL1pro and PL2pro, cleavage sites=white arrow)and adenosine diphosphate-ribose 1″-phosphatase (ADRP, X domain)activity. The PL2pro is also an interferon antagonist. Nsp5 is the mainprotease or 3C-like protease (3CLpro, cleavage site=black arrow). Nsp3,nsp4 and nsp6 contain transmembrane domains (TM) and function asmembrane anchor proteins. Nsp7 and nsp8 form a hexadecameric complexwith a central channel for binding to the double-stranded RNA andinitiating RNA synthesis. Nsp9 is a single-stranded RNA-binding protein(RBP). Nsp10 is a co-factor (activator) of nsp14 and nsp16. The nsp14has 3′-5′ exonuclease (ExoN) at the N-terminal and 7-methyltransferase(7MT) at the C-terminal. The nsp16 acts as the 2′-O-methyltransferase(2′-O-MT). The interactions between nsp10, nsp14 and nsp16 plays acrucial role in replication fidelity and methylation of the virus. Thensp11 function is not fully understood yet. Nsp12 is the viralRNA-dependent RNA polymerase (RdRP). Nsp13 harbors a zinc-binding domain(ZBD) and viral helicase. Nsp13 also has NTPase and RNA 5′triphosphatase activity. Nsp15 contains a motif known as nidoviraluridylate-specific endoribonuclease (NendoU). The S protein functions asthe virus attachment protein interacting with the cell receptor.Additionally, the S protein is postulated to harbor neutralizationepitopes and is also associated with viral virulence/attenuation. The Mprotein and E protein play a pivotal role in viral assembly. The Nprotein binds to the viral genome RNA and packs into the nucleocapsid.The N protein has also been shown to antagonize interferon-n production.The accessory protein encoded by ORF3 may be associated with cellculture adaptation and may also have an influence on cell cycle andsubcellular structure.

FIG. 13 is an image showing IFA antibody testing of antisera against theU.S. PEDV prototype and S-INDEL-variant strains. The average IFAantibody titers are shown at the top and the number of IFA antibodypositive samples is shown at the bottom. Pro antisera: antiseracollected from the U.S. PEDV prototype strain-inoculated pigs; Varantisera: antisera collected from the U.S. PEDV S-INDEL-variantstrain-inoculated pigs; Neg antisera: antisera collected from negativecontrol pigs; Pro IFA: the U.S. PEDV prototype strain-based IFA; VarIFA: the U.S. PEDV S-INDEL-variant strain-based IFA

FIG. 14A-14C show testing of antisera against the U.S. PEDV prototypeand S-INDEL-variant strains by ProWV ELISA (FIG. 14A), ProS1 ELISA (FIG.14B) and VarS1 ELISA (FIG. 14C). For each assay, the solid black lineindicates the S/P ratio above which the sample was positive; the dotblack line indicates the S/P ratio below which the sample was negative;samples with S/P ratios between the solid and dot black line weresuspect. ProWV ELISA: the U.S. PEDV prototype strain whole virus-basedELISA; ProS1 ELISA: the U.S. PEDV prototype strain S1-based ELISA; VarS1ELISA: the U.S. PEDV S-INDEL-variant strain S1-based ELISA.

FIG. 15 shows virus neutralization antibody testing of antisera againstthe U.S. PEDV prototype and S-INDEL-variant strains. The average VNantibody titers are shown at the top and the number of VN antibodypositive samples is shown at the bottom. Pro VN: the U.S. PEDV prototypestrain-based VN; Var VN: the U.S. PEDV S-INDEL-variant strain-based VN.

FIG. 16 show a table comparing the nucleotide and amino acid changes ofU.S. prototype PEDV isolate USA/IN19338/2013 during serial passages incell culture.

FIG. 17 shows the growth curve of the U.S. PEDV prototype isolateUSA/IN19338/2013 P7, P25, P50, P75 and P100 in Vero cells.

FIG. 18 is a graph showing virus shedding in rectal swabs of variousinoculation groups during 0-7 days post inoculation (DPI). Differentletters indicate significant differences.

FIG. 19 shows small intestine microscopic lesion severity scores invarious groups of piglets inoculated with the PEDV USA/IN19338/2013 P7,P25, P50, P75 and P100 at 3 days post inoculation. Labels without thesame letters indicate significant differences, for example A and B havesignificant differences but A and AB have no significant differences.

FIG. 20 shows the average villus-height-to-crypt-depth ratios of smallintestines in various groups of piglets inoculated with the PEDVUSA/IN19338/2013 P7, P25, P50, P75 and P100 at 3 days post inoculation.Different letters indicate significant differences.

FIG. 21 shows the average immunohistochemistry scores in smallintestines, ceca and colons in various groups of piglets inoculated withthe PEDV USA/IN19338/2013 P7, P25, P50, P75 and P100 at 3 days postinoculation.

FIG. 22A-22C shows sequences according to exemplary embodiments of theinvention. FIG. 22A shows the sequence of 2014020697-P8R1 passage 8,lineage 2 ORF1a/1b polyprotein (SEQ ID NO: 17); 2014020697-P8R1 passage8, lineage 2 spike protein (SEQ ID NO: 18), 2014020697-P8R1 passage 8,lineage 2 ORF 3 protein (truncated) (SEQ ID NO: 19), 2014020697-P8R1passage 8, lineage 2 envelope protein (SEQ ID NO: 20), 2014020697-P8R1passage 8, lineage 2 membrane protein (SEQ ID NO: 21), and2014020697-P8R1 passage 8, lineage 2 nucleocapsid protein (SEQ ID NO:22). FIG. 22B shows the 2014020697-P18R1 passage 18, lineage 2 clone G8bORF1a/1b polyprotein (SEQ ID NO: 23), 2014020697-P18R1 passage 18,lineage 2 clone G8b spike protein (SEQ ID NO: 24), 2014020697-P18R1passage 18, lineage 2 clone G8b ORF3 protein (truncated) (SEQ ID NO:25), 2014020697-P18R1 passage 18, lineage 2 clone G8b envelope protein(SEQ ID NO: 26), 2014020697-P18R1 passage 18, lineage 2 clone G8bmembrane protein (SEQ ID NO: 27) and 2014020697-P18R1 passage 18,lineage 2 clone G8b nucleocapsid protein (SEQ ID NO: 28). FIG. 22C is2014020697-P18R1 passage 18, lineage 2 clone F6a ORF1a/1b polyprotein(SEQ ID NO: 29); 2014020697-P18R1 passage 18, lineage 2 clone F6a spikeprotein (SEQ ID NO: 30), 2014020697-P18R1 passage 18, lineage 2 cloneF6a ORF 3 protein (truncated) (SEQ ID NO: 31), 2014020697-P18R1 passage18, lineage 2 clone F6a envelope protein (SEQ ID NO: 32),2014020697-P18R1 passage 18, lineage 2 clone F6a membrane protein (SEQID NO: 33), and 2014020697-P18R1 passage 18, lineage 2 clone F6anucleocapsid protein (SEQ ID NO: 34).

FIG. 23A-23C shows sequences according to exemplary embodiments theinvention. FIG. 23A shows the genomic nucleotide sequence for2014020697-P8R1 passage 8, lineage 2 (SEQ ID NO:35), FIG. 23B shows thenucleotide sequence for 2014020697-P18R1 passage 18, lineage 2 clone G8bcomplete genome (SEQ ID NO: 36); FIG. 23C shows 2014020697-P18R1 passage18, lineage 2 clone F6a complete genome nucleotide sequence (SEQ ID NO:37).

FIGS. 24A-24C are a chart showing the INDEL sequence comparison forvarious passages.

FIGS. 25A to 25H are the genomic nucleotide sequence of 2014020697-P45passage 45, lineage 1 (SEQ ID NO:39).

FIGS. 26A to 26JJJ are a comparison of the genomic nucleotide sequencesof 2014020697-P5 passages 5, lineage 1 (P5) (SEQ ID NO:8),2014020697-P7R1 passage 7, lineage 2 (SEQ ID NO:15), 2014020697-P8R1passage 8, lineage 2 (SEQ ID NO:35), 2014020697-P18R1 passage 18,lineage 2 clone G8b (SEQ ID NO:36), 2014020697-P18R1 passage 18, lineage2 clone F6a (SEQ ID NO:37) and 2014020697-P45 passage 45, lineage 1 (SEQID NO:39).

FIGS. 27A-27C show sequences according to exemplary embodiments of theinvention. The amino acid sequences of 2014020697-P45 passage 45,lineage 1 ORF1a/1b polyprotein (SEQ ID NO: 40); 2014020697-P45 passage45, lineage 1 spike protein (SEQ ID NO:41); 2014020697-P45 passage 45,lineage 1 ORF3 protein (SEQ ID NO:42); 2014020697-P45 passage 45,lineage 1 envelope protein (SEQ ID NO:43); 2014020697-P45 passage 45,lineage 1 membrane protein (SEQ ID NO:44); and 2014020697-P45 passage45, lineage 1 nucleocapsid protein (SEQ ID NO:45) are shown.

FIG. 28A-28B shows fecal virus shedding titers. FIG. 28A shows fecalvirus shedding titers on each time point of each group with upperstandard error bars shown. FIG. 28B shows statistic analysis of overallvirus shedding titer in rectal swabs on D0-D28 and D28-D56. DuringD0-D28, the N/N, N/V and N/P groups received the same inoculum and wereanalyzed as the same treatment, similarly for the V/V and V/P groups,and P/V and P/P groups. Different letters indicate statisticallysignificant difference in virus shedding amount between groups.

FIG. 29A-29B Macro-pathological scores, FIG. 29A is the group meanmacro-pathological scores at D4 (4 days post the 1^(st) inoculation)with the standard error bars shown. FIG. 29B is the group meanmacro-pathological scores at D34 (6 days post the challenge at D28) withthe standard error bars shown. Small intestine, cecum, and colon contentscores as well as the gross lesion scores of small intestine, cecum andcolon organs were shown. Different letters indicate statisticallysignificant differences observed with the same parameter between groups.Yellow highlighted are statistics of V-strain challenged pigs, and greenhighlighted are statistics of P-strain challenged pigs.

FIG. 30A-30B shows the histopathology measurement at D4. FIG. 30A showsthe group mean of villus height/crypt depth ratios of distal jejunum andileum with the standard error bars shown. FIG. 30B shows the group meanof PEDV IHC scores in distal jejunum, ileum, cecum, and colon with thestandard error bars shown. Different letters indicate statisticallysignificant differences observed in the same tissue between groups.

FIG. 31A-31B shows the histopathology measurement at D34. FIG. 31A showthe group mean of villus height/crypt depth ratios of distal jejunum andileum with the standard error bars shown. FIG. 31B shows the group meanof PEDV IHC scores in distal jejunum, ileum, cecum, and colon with thestandard error bars shown. Different letters indicate statisticallysignificant differences observed in the same tissue between groups.Yellow highlighted are statistics of V-strain challenged pigs, and greenhighlighted are statistics of P-strain challenged pigs.

FIG. 32A-32B shows the serum IFA antibody titers against either P-strainvirus or V-strain virus. FIG. 32A shows the group mean of serum IFAtiters against P-strain virus with the standard error bars shown. FIG.32B shows the group mean of serum IFA titers against V-strain virus withthe standard error bars shown.

FIG. 33A-33B shows the serum VN antibody titers against either P-strainvirus or V-strain virus. FIG. 33A shows the group mean of serum VNtiters against P-strain virus with the standard error bars shown. FIG.33B shows the group mean of serum VN titers against V-strain virus withthe standard error bars shown.

FIGS. 34A-34B show the nucleotide and amino acid changes of PEDV isolateIL20697/2014 during serial passages in cell culture.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and introductory matters are applicable in thespecification.

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicate otherwise.The word “or” means any one member of a particular list and alsoincludes any combination of members of that list.

The term “adjuvant” refers to a compound that enhances the effectivenessof the vaccine, and may be added to the formulation that includes theimmunizing agent. Adjuvants provide enhanced immune response even afteradministration of only a single dose of the vaccine. Adjuvants mayinclude, for example, muramyl dipeptides, pyridine, aluminum hydroxide,dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-wateremulsions, saponins, cytokines, and other substances known in the art.Examples of suitable adjuvants are described in U.S. Patent ApplicationPublication No. US2004/0213817 A1. “Adjuvanted” refers to a compositionthat incorporates or is combined with an adjuvant.

“Antibodies” refers to polyclonal and monoclonal antibodies, chimeric,and single chain antibodies, as well as Fab fragments, including theproducts of a Fab or other immunoglobulin expression library. Withrespect to antibodies, the term, “immunologically specific” refers toantibodies that bind to one or more epitopes of a protein of interest,but which do not substantially recognize and bind other molecules in asample containing a mixed population of antigenic biological molecules.

An “attenuated” PEDV as used herein refers to a PEDV which is capable ofinfecting and/or replicating in a susceptible host, but isnon-pathogenic or less-pathogenic to the susceptible host. For example,the attenuated virus may cause no observable/detectable clinicalmanifestations, or less clinical manifestations, or less severe clinicalmanifestations, or exhibit a reduction in virus replication efficiencyand/or infectivity, as compared with the related field isolated strains.The clinical manifestations of PEDV infection can include, withoutlimitation, clinical diarrhea, vomiting, lethargy, loss of condition anddehydration.

An “epitope” is an antigenic determinant that is immunologically activein the sense that once administered to the host, it is able to evoke animmune response of the humoral (B cells) and/or cellular type (T cells).These are particular chemical groups or peptide sequences on a moleculethat are antigenic. An antibody specifically binds a particularantigenic epitope on a polypeptide. In the animal most antigens willpresent several or even many antigenic determinants simultaneously. Sucha polypeptide may also be qualified as an immunogenic polypeptide andthe epitope may be identified as described further.

The term “immunogenic fragment” as used herein refers to a polypeptideor a fragment of a polypeptide, or a nucleotide sequence encoding thesame which comprises an allele-specific motif, an epitope or othersequence such that the polypeptide or the fragment will bind an MHCmolecule and induce a cytotoxic T lymphocyte (“CTL”) response, and/or aB cell response (for example, antibody production), and/or T-helperlymphocyte response, and/or a delayed type hypersensitivity (DTH)response against the antigen from which the immunogenic polypeptide orthe immunogenic fragment is derived. A DTH response is an immunereaction in which T cell-dependent macrophage activation andinflammation cause tissue injury. A DTH reaction to the subcutaneousinjection of antigen is often used as an assay for cell-mediatedimmunity.

With the term “induction of an immunoprotective response” is meant a(humoral and/or cellular) immune response that reduces or eliminates oneor more of the symptoms of disease, i.e. clinical signs, lesions,bacterial excretion and bacterial replication in tissues in the infectedsubject compared to a healthy control. Preferably said reduction insymptoms is statistically significant when compared to a control.

An “infectious DNA molecule”, for purposes of the present invention, isa DNA molecule that encodes the necessary elements for viralreplication, transcription, and translation into a functional virion ina suitable host cell.

The term “isolated” is used to indicate that a cell, peptide or nucleicacid is separated from its native environment. Isolated peptides andnucleic acids may be substantially pure, i.e. essentially free of othersubstances with which they may bound in nature.

For purposes of the present invention, the nucleotide sequence of asecond polynucleotide molecule (either RNA or DNA) is “homologous” tothe nucleotide sequence of a first polynucleotide molecule, or has“identity” to said first polynucleotide molecule, where the nucleotidesequence of the second polynucleotide molecule encodes the samepolyaminoacid as the nucleotide sequence of the first polynucleotidemolecule as based on the degeneracy of the genetic code, or when itencodes a polyaminoacid that is sufficiently similar to thepolyaminoacid encoded by the nucleotide sequence of the firstpolynucleotide molecule so as to be useful in practicing the presentinvention. Homologous polynucleotide sequences also refers to sense andanti-sense strands, and in all cases to the complement of any suchstrands. For purposes of the present invention, a polynucleotidemolecule is useful in practicing the present invention, and is thereforehomologous or has identity, where it can be used as a diagnostic probeto detect the presence of PEDV or viral polynucleotide in a fluid ortissue sample of an infected pig, e.g. by standard hybridization oramplification techniques. Generally, the nucleotide sequence of a secondpolynucleotide molecule is homologous to the nucleotide sequence of afirst polynucleotide molecule if it has at least about 70% nucleotidesequence identity to the nucleotide sequence of the first polynucleotidemolecule as based on the BLASTN algorithm (National Center forBiotechnology Information, otherwise known as NCBI, (Bethesda, Md., USA)of the United States National Institute of Health). In a specificexample for calculations according to the practice of the presentinvention, reference is made to BLASTP 2.2.6 [Tatusova TA and TL Madden,“BLAST 2 sequences—a new tool for comparing protein and nucleotidesequences.” (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 0.1, and the“blosum62” scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad.Sci. USA 325 89:10915-10919. 1992). The percent identity is thencalculated as: Total number of identical matches X 100/divided by thelength of the longer sequence+number of gaps introduced into the longersequence to align the two sequences.

Preferably, a homologous nucleotide sequence has at least about 75%nucleotide sequence identity, even more preferably at least about 80%,85%, 90% and 95% nucleotide sequence identity. Since the genetic code isdegenerate, a homologous nucleotide sequence can include any number of“silent” base changes, i.e. nucleotide substitutions that nonethelessencode the same amino acid.

A homologous nucleotide sequence can further contain non-silentmutations, i.e. base substitutions, deletions, or additions resulting inamino acid differences in the encoded polyaminoacid, so long as thesequence remains at least about 70% identical to the polyaminoacidencoded by the first nucleotide sequence or otherwise is useful forpracticing the present invention. In this regard, certain conservativeamino acid substitutions may be made which are generally recognized notto inactivate overall protein function: such as in regard of positivelycharged amino acids (and vice versa), lysine, arginine and histidine; inregard of negatively charged amino acids (and vice versa), aspartic acidand glutamic acid; and in regard of certain groups of neutrally chargedamino acids (and in all cases, also vice versa), (1) alanine and serine,(2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4)glycine and proline, (5) isoleucine, leucine and valine, (6) methionine,leucine and isoleucine, (7) phenylalanine, methionine, leucine, andtyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10)and for example tyrosine, tyrptophan and phenylalanine. Amino acids canbe classified according to physical properties and contribution tosecondary and tertiary protein structure. A conservative substitution isthus recognized in the art as a substitution of one amino acid foranother amino acid that has similar properties, and exemplaryconservative substitutions may be found in WO 97/09433, page 10,published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996.Alternatively, conservative amino acids can be grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp. 71-77). Protein sequences can be aligned using both VectorNTI Advance 11.5 and CLUSTAL 2.1 multiple sequence alignment. As usedherein the recitation of a particular amino acid or nucleotide sequenceshall include all silent mutations with respect to nucleic acid sequenceand any and all conservatively modified variants with respect to aminoacid sequences.

Homologous nucleotide sequences can be determined by comparison ofnucleotide sequences, for example by using BLASTN, above. Alternatively,homologous nucleotide sequences can be determined by hybridization underselected conditions. For example, the nucleotide sequence of a secondpolynucleotide molecule is homologous to SEQ ID NO:1 (or any otherparticular polynucleotide sequence) if it hybridizes to the complementof SEQ ID NO:1 under moderately stringent conditions, e.g.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at42° C. (see Ausubel et al editors, Protocols in Molecular Biology, Wileyand Sons, 1994, pp. 6.0.3 to 6.4.10), or conditions which will otherwiseresult in hybridization of sequences that encode a PEDV virus as definedbelow. Modifications in hybridization conditions can be empiricallydetermined or precisely calculated based on the length and percentage ofguanosine/cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook, et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.

In another embodiment, a second nucleotide sequence is homologous to SEQID NO: 1 (or any other sequence of the invention) if it hybridizes tothe complement of SEQ ID NO: 1 under highly stringent conditions, e.g.hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C., and washing in 0.1×SSC/0.1% SDS at 68° C., as is known in theart.

It is furthermore to be understood that the isolated polynucleotidemolecules and the isolated RNA molecules of the present inventioninclude both synthetic molecules and molecules obtained throughrecombinant techniques, such as by in vitro cloning and transcription.

It should be noted that many of the vaccine-capable attenuated PEDVviruses of the present invention contain substantial deletions of ORF3protein, resulting from attenuating mutations in the ORF3 nucleotidesequence, that cause substantial internal deletions and/or mosttypically the appearance of truncated translation resultant fromframeshifting and the appearance of stop codons. It should therefore benoted that within the practice of the present invention, alignment andpercent identity calculations and stated identity results (whether ornucleotide or amino acid sequences) can be calculated with or withoutreference to deleted ORF3 sequences.

“Mammals” include any warm-blooded vertebrates of the Mammalia class,including humans.

A “pharmaceutically acceptable carrier” means any conventionalpharmaceutically acceptable carrier, vehicle, or excipient that is usedin the art for production and administration of vaccines.Pharmaceutically acceptable carriers are typically non-toxic, inert,solid or liquid carriers.

The terms “porcine” and “swine” are used interchangeably herein andrefer to any animal that is a member of the family Suidae such as, forexample, a pig.

A “susceptible” host as used herein refers to a cell or an animal thatcan be infected by PEDV. When introduced to a susceptible animal, anattenuated PEDV may also induce an immunological response against thePEDV or its antigen, and thereby render the animal immunity against PEDVinfection.

The term “vaccine” refers to an antigenic preparation used to produceimmunity to a disease, in order to prevent or ameliorate the effects ofinfection. Vaccines are typically prepared using a combination of animmunologically effective amount of an immunogen together with anadjuvant effective for enhancing the immune response of the vaccinatedsubject against the immunogen.

Vaccine formulations will contain a “therapeutically effective amount”of the active ingredient, that is, an amount capable of eliciting aninduction of an immunoprotective response in a subject to which thecomposition is administered. In the treatment and prevention of PEDVdisease, for example, a “therapeutically effective amount” wouldpreferably be an amount that enhances resistance of the vaccinatedsubject to new infection and/or reduces the clinical severity of thedisease. Such protection will be demonstrated by either a reduction orlack of symptoms normally displayed by a subject infected with PEDV, aquicker recovery time and/or a lowered count of virus particles.Vaccines can be administered prior to infection, as a preventativemeasure against PEDV. Alternatively, vaccines can be administered afterthe subject already has contracted a disease. Vaccines given afterexposure to PEDV may be able to attenuate the disease, triggering asuperior immune response than the natural infection itself.

For the purpose of the practice of all aspects of the invention, it iswell known to those skilled in the art that there is no absoluteimmunological boundary in immunological assays in regard of animals thatare seronegative for exposure to a particular antigen or pathogen, andthose that are seropositive (having been exposed to a vaccine orpathogen). Nonetheless, those skilled in the art would recognize that inserum neutralization assays, seropositive animals would generally bedetected at least up to a 1:1000 serum dilution, whereas a seronegativeanimal would be expected not to neutralize at a higher dilution thatabout 1:20 or 1:10.

Vaccine Formulations/Immunogenic Compositions

The invention also relates to an immunogenic composition, suitable to beused as a vaccine, which comprises a variant PEDV strain according tothe invention. The immunogenic compositions according to the inventionelicit a specific humoral immune response toward the PEDV comprisingneutralizing antibodies.

The preferred immunogenic compositions based upon the variant strainsdisclosed herein can provide live, attenuated viruses which exhibit highimmunogenicity while at the same time not producing dangerous pathogenicor lethal effects.

The immunogenic compositions of this invention are not, however,restricted to any particular type or method of preparation. Theseinclude, but are not limited to, infectious DNA vaccines (i.e., usingplasmids, vectors or other conventional carriers to directly inject DNAinto pigs), live vaccines, modified live vaccines, inactivated vaccines,subunit vaccines, attenuated vaccines, genetically engineered vaccines,etc. These vaccines are prepared by standard methods known in the art.

The present invention preferably includes vaccine compositionscomprising a live, attenuated variant PEDV of the invention and apharmaceutically acceptable carrier. As used herein, the expression“live, attenuated PEDV of the invention” encompasses any live,attenuated PEDV strain that includes one or more of the variationsdescribed herein. The pharmaceutically acceptable carrier can be, e.g.,water, a stabilizer, a preservative, culture medium, or a buffer.Vaccine formulations comprising the attenuated PEDV of the invention canbe prepared in the form of a suspension or in a lyophilized form or,alternatively, in a frozen form. If frozen, glycerol or other similaragents may be added to enhance stability when frozen. The advantages oflive attenuated vaccines, in general, include the presentation of allthe relevant immunogenic determinants of an infectious agent in itsnatural form to the host's immune system, and the need for relativelysmall amounts of the immunizing agent due to the ability of the agent tomultiply in the vaccinated host.

Attenuation of the virus for a live vaccine, so that it isinsufficiently pathogenic to substantially harm the vaccinated targetanimal, may be accomplished by known procedures, including preferably byserial passaging. The following references provide various generalmethods for attenuation of coronaviruses, and are suitable forattenuation or further attenuation of any of the strains useful in thepractice of the present invention: B. Neuman et al., Journal ofVirology, vol. 79, No. 15, pp. 9665-9676, 2005; J. Netland et al.,Virology, v 399(1), pp. 120-128, 2010; Y-P Huang et al., “Sequencechanges of infectious bronchitis virus isolates in the 3′ 7.3 kb of thegenome after attenuating passage in embryonated eggs, Avian Pathology,v. 36 (1), (Abstract), 2007; and S. Hingley et al., Virology, v. 200(1)1994, pp. 1-10; see U.S. Pat. No. 3,914,408; and Ortego et al.,Virology, vol. 308 (1), pp. 13-22, 2003.

Additional genetically engineered vaccines, which are desirable in thepresent invention, are produced by techniques known in the art. Suchtechniques involve, but are not limited to, further manipulation ofrecombinant DNA, modification of or substitutions to the amino acidsequences of the recombinant proteins and the like.

Genetically engineered vaccines based on recombinant DNA technology aremade, for instance, by identifying alternative portions of the viralgene encoding proteins responsible for inducing a stronger immune orprotective response in pigs (e.g., proteins derived from M, GP2, GP3,GP4, or GP5, etc.). Various subtypes or isolates of the viral proteingenes can be subjected to the DNA-shuffling method. The resultingheterogeneous chimeric viral proteins can be used broad protectingsubunit vaccines. Alternatively, such chimeric viral genes orimmuno-dominant fragments can be cloned into standard protein expressionvectors, such as the baculovirus vector, and used to infect appropriatehost cells (see, for example, O'Reilly et al., “Baculovirus ExpressionVectors: A Lab Manual,” Freeman & Co., 1992). The host cells arecultured, thus expressing the desired vaccine proteins, which can bepurified to the desired extent and formulated into a suitable vaccineproduct.

If the clones retain any undesirable natural abilities of causingdisease, it is also possible to pinpoint the nucleotide sequences in theviral genome responsible for any residual virulence, and geneticallyengineer the virus avirulent through, for example, site-directedmutagenesis. Site-directed mutagenesis is able to add, delete or changeone or more nucleotides (see, for instance, Zoller et al., DNA3:479-488, 1984). An oligonucleotide is synthesized containing thedesired mutation and annealed to a portion of single stranded viral DNA.The hybrid molecule, which results from that procedure, is employed totransform bacteria. Then double-stranded DNA, which is isolatedcontaining the appropriate mutation, is used to produce full-length DNAby ligation to a restriction fragment of the latter that is subsequentlytransfected into a suitable cell culture. Ligation of the genome intothe suitable vector for transfer may be accomplished through anystandard technique known to those of ordinary skill in the art.Transfection of the vector into host cells for the production of viralprogeny may be done using any of the conventional methods such ascalcium-phosphate or DEAE-dextran mediated transfection,electroporation, protoplast fusion and other well-known techniques(e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” ColdSpring Harbor Laboratory Press, 1989). The cloned virus then exhibitsthe desired mutation. Alternatively, two oligonucleotides can besynthesized which contain the appropriate mutation. These may beannealed to form double-stranded DNA that can be inserted in the viralDNA to produce full-length DNA.

An immunologically effective amount of the vaccines of the presentinvention is administered to a pig in need of protection against viralinfection. The immunologically effective amount or the immunogenicamount that inoculates the pig can be easily determined or readilytitrated by routine testing. An effective amount is one in which asufficient immunological response to the vaccine is attained to protectthe pig exposed to the PEDV virus. Preferably, the pig is protected toan extent in which one to all of the adverse physiological symptoms oreffects of the viral disease are significantly reduced, ameliorated ortotally prevented.

Vaccines of the present invention can be formulated following acceptedconvention to include acceptable carriers for animals, such as standardbuffers, stabilizers, diluents, preservatives, and/or solubilizers, andcan also be formulated to facilitate sustained release. Diluents includewater, saline, dextrose, ethanol, glycerol, and the like. Additives forisotonicity include sodium chloride, dextrose, mannitol, sorbitol, andlactose, among others. Stabilizers include albumin, among others. Othersuitable vaccine vehicles and additives, including those that areparticularly useful in formulating modified live vaccines, are known orwill be apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention may further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Non-limiting examples of adjuvants that can beused in the vaccine of the present invention include the RIBI adjuvantsystem (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminumhydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block copolymer(CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.),SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil Aor other saponin fraction, monophosphoryl lipid A, ionicpolysaccharides, and Avridine lipid-amine adjuvant. Non-limitingexamples of oil-in-water emulsions useful in the vaccine of theinvention include modified SEAM62 and SEAM 1/2 formulations. ModifiedSEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma),1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100mg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is anoil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 mg/mlQuil A, and 50 mg/ml cholesterol. Other immunomodulatory agents that canbe included in the vaccine include, e.g., one or more interleukins,interferons, or other known cytokines.

Additional adjuvant systems permit for the combination of both T-helperand B-cell epitopes, resulting in one or more types of covalent T-Bepitope linked structures, with may be additionally lipidated, such asthose described in WO2006/084319, WO2004/014957, and WO2004/014956.

In a preferred embodiment of the present invention, ORFI PEDV protein,or other PEDV proteins or fragments thereof, is formulated with 5%AMPHIGEN® as discussed hereinafter.

Adjuvant Components

The vaccine compositions of the invention may or may not includeadjuvants. In particular, as based on an orally infective virus, themodified live vaccines of the invention may be used adjuvant free, witha sterile carrier. Adjuvants that may be used for oral administrationinclude those based on CT-like immune modulators (rmLT, CT-B, i.e.recombinant-mutant heat labile toxin of E. coli, Cholera toxin-Bsubunit); or via encapsulation with polymers and alginates, or withmucoadhesives such as chitosan, or via liposomes. A preferred adjuvantedor non adjuvanted vaccine dose at the minimal protective dose throughvaccine release may provide between approximately 10 and approximately10⁶ log₁₀TCID₅₀ of virus per dose, or higher. “TCID50” refers to “tissueculture infective dose” and is defined as that dilution of a virusrequired to infect 50% of a given batch of inoculated cell cultures.Various methods may be used to calculate TCID50, including theSpearman-Karber method which is utilized throughout this specification.For a description of the Spearman-Karber method, see B. W. Mahy & H. O.Kangro, Virology Methods Manual, p. 25-46 (1996). Adjuvants, if present,may be provided as emulsions, more commonly if non-oral administrationis selected, but should not decrease starting titer by more than 0.7logs (80% reduction).

In one example, adjuvant components are provided from a combination oflecithin in light mineral oil, and also an aluminum hydroxide component.Details concerning the composition and formulation of Amphigen® (asrepresentative lecithin/mineral oil component) are as follows.

A preferred adjuvanted may be provided as a 2 ML dose in a bufferedsolution further comprising about 5% (v/v) Rehydragel® (aluminumhydroxide gel) and “20% Amphigen”® at about 25% final (v/v). Amphigen®is generally described in U.S. Pat. No. 5,084,269 and provides de-oiledlecithin (preferably soy) dissolved in a light oil, which is thendispersed into an aqueous solution or suspension of the antigen as anoil-in-water emulsion. Amphigen has been improved according to theprotocols of U.S. Pat. No. 6,814,971 (see columns 8-9 thereof) toprovide a so-called “20% Amphigen” component for use in the finaladjuvanted vaccine compositions of the present invention. Thus, a stockmixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, KarnsCity, Pa.) is diluted 1:4 with 0.63% phosphate buffered saline solution,thereby reducing the lecithin and DRAKEOL components to 2% and 18%respectively (i.e. 20% of their original concentrations). Tween 80 andSpan 80 surfactants are added to the composition, with representativeand preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v)Span 80, wherein the Span is originally provided in the stock DRAKEOLcomponent, and the Tween is originally provided from the buffered salinecomponent, so that mixture of the saline and DRAKEOL components resultsin the finally desired surfactant concentrations. Mixture of theDRAKEOL/lecithin and saline solutions can be accomplished using anIn-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son,Hauppauge, N.Y., USA.

The vaccine composition also includes Rehydragel® LV (about 2% aluminumhydroxide content in the stock material), as additional adjuvantcomponent (available from Reheis, N.J., USA, and ChemTrade Logistics,USA). With further dilution using 0.63% PBS, the final vaccinecomposition contains the following compositional amounts per 2 ML dose;5% (v/v) Rehydragel® LV; 25% (v/v) of “20% Amphigen”, i.e. it is further4-fold diluted); and 0.01% (w/v) of merthiolate.

As is understood in the art, the order of addition of components can bevaried to provide the equivalent final vaccine composition. For example,an appropriate dilution of virus in buffer can be prepared. Anappropriate amount of Rehydragel® LV (about 2% aluminum hydroxidecontent) stock solution can then be added, with blending, in order topermit the desired 5% (v/v) concentration of Rehydragel® LV in theactual final product. Once prepared, this intermediate stock material iscombined with an appropriate amount of “20% Amphigen” stock (asgenerally described above, and already containing necessary amounts ofTween 80 and Span 80) to again achieve a final product having 25% (v/v)of “20% Amphigen”. An appropriate amount of 10% merthiolate can finallybe added.

The vaccinate compositions of the invention permit variation in all ofthe ingredients, such that the total dose of antigen may be variedpreferably by a factor of 100 (up or down) compared to the antigen dosestated above, and most preferably by a factor of 10 or less (up ordown). Similarly, surfactant concentrations (whether Tween or Span) maybe varied by up to a factor of 10, independently of each other, or theymay be deleted entirely, with replacement by appropriate concentrationsof similar materials, as is well understood in the art.

Rehydragel® concentrations in the final product may be varied, first bythe use of equivalent materials available from many other manufacturers(i.e. Alhydrogel®, Brenntag; Denmark), or by use of additionalvariations in the Rehydragel® line of products such as CG, HPA or HS.Using LV as an example, final useful concentrations thereof includingfrom 0% to 20%, with 2-12% being more preferred, and 4-8% being mostpreferred, Similarly, the although the final concentration of Amphigen(expressed as % of “20% Amphigen”) is preferably 25%, this amount mayvary from 5-50%, preferably 20-30% and is most preferably about 24-26%.

According to the practice of the invention, the oil used in the adjuvantformulations of the instant invention is preferably a mineral oil. Asused herein, the term “mineral oil” refers to a mixture of liquidhydrocarbons obtained from petrolatum via a distillation technique. Theterm is synonymous with “liquefied paraffin”, “liquid petrolatum” and“white mineral oil.” The term is also intended to include “light mineraloil,” i.e., oil which is similarly obtained by distillation ofpetrolatum, but which has a slightly lower specific gravity than whitemineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18thEdition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and1323). Mineral oil can be obtained from various commercial sources, forexample, J. T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland,Ohio). Preferred mineral oil is light mineral oil commercially availableunder the name DRAKEOL®.

Typically, the oily phase is present in an amount from 50% to 95% byvolume; preferably, in an amount of greater than 50% to 85%; morepreferably, in an amount from greater than 50% to 60%, and morepreferably in the amount of greater than 50-52% v/v of the vaccinecomposition. The oily phase includes oil and emulsifiers (e.g., SPAN®80, TWEEN® 80 etc), if any such emulsifiers are present.

Non-natural, synthetic emulsifiers suitable for use in the adjuvantformulations of the present invention also include sorbitan-basednon-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants(commercially available under the name SPAN® or ARLACEL®), fatty acidesters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol estersof fatty acids from sources such as castor oil (EMULFOR®);polyethoxylated fatty acid (e.g., stearic acid available under the nameSIMULSOL® M-53), polyethoxylated isooctylphenol/formaldehyde polymer(TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®);polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethyleneisooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants arethe surfactants available under the name SPAN® and TWEEN®, such asTWEEN®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN®-80(sorbitan monooleate). Generally speaking, the emulsifier(s) may bepresent in the vaccine composition in an amount of 0.01% to 40% byvolume, preferably, 0.1% to 15%, more preferably 2% to 10%.

In an alternative embodiment of the invention, the final vaccinecomposition contains SP-Oil® and Rehydragel® LV as adjuvants (or otherRehydragel® or Alhydrogel® products), with preferable amounts beingabout 5-20% SP-Oil (v/v) and about 5-15% Rehydragel LV (v/v), and with5% and 12%, respectively, being most preferred amounts. In this regardit is understood that % Rehydragel refers to percent dilution from thestock commercial product. (SP-Oil® is a fluidized oil emulsion withincludes a polyoxyethylene-polyoxypropylene block copolymer (Pluronic®L121, BASF Corporation, squalene, polyoxyethylene sorbitan monooleate(Tween®80, ICI Americas) and a buffered salt solution.)

It should be noted that the present invention may also be successfullypracticed using wherein the adjuvant component is only Amphigen®.

In another embodiment of the invention, the final vaccine compositioncontains TXO as an adjuvant; TXO is generally described in WO2015/042369. All TXO compositions disclosed therein are useful in thepreparation of vaccines of the invention. In TXO, the immunostimulatoryoligonucleotide (“T”), preferably an ODN, preferably containing apalindromic sequence, and optionally with a modified backbone, ispresent in the amount of 0.1 to 5 ug per 50 ul of the vaccinecomposition (e.g., 0.5-3 ug per 50 ul of the composition, or morepreferably 0.09-0.11 ug per 50 ul of the composition). A preferredspecies thereof is SEQ ID NO: 8 as listed (page 17) in the WO2015/042369publication (PCT/US2014/056512). The polycationic carrier (“X”) ispresent in the amount of 1-20 ug per 50 ul (e.g., 3-10 ug per 50 ul, orabout 5 ug per 50 ul). Light mineral oil (“O”) is also a component ofthe TXO adjuvant.

In certain embodiments, TXO adjuvants are prepared as follows:

a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in lightmineral oil. The resulting oil solution is sterile filtered;

b) The immunostimulatory oligonucleotide, Dextran DEAE andPolyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase,thus forming the aqueous solution; and

c) The aqueous solution is added to the oil solution under continuoushomogenization thus forming the adjuvant formulation TXO.

All the adjuvant compositions of the invention can be used with any ofthe PEDV strains and isolates covered by the present Specification.

Additional adjuvants useful in the practice of the invention includePrezent-A (see generally United States published patent applicationUS20070298053; and “QCDCRT” or “QCDC”-type adjuvants (see generallyUnited States published patent application US20090324641.

Excipients

The immunogenic and vaccine compositions of the invention can furthercomprise pharmaceutically acceptable carriers, excipients and/orstabilizers (see e.g. Remington: The Science and practice of Pharmacy,2005, Lippincott Williams), in the form of lyophilized formulations oraqueous solutions. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations, and maycomprise buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas Mercury((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL),octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG), TWEEN or PLURONICS.

Vaccines of the present invention can optionally be formulated forsustained release of the virus, infectious DNA molecule, plasmid, orviral vector of the present invention. Examples of such sustainedrelease formulations include virus, infectious DNA molecule, plasmid, orviral vector in combination with composites of biocompatible polymers,such as, e.g., poly (lactic acid), poly (lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including A. Domb et al., 1992,Polymers for Advanced Technologies 3: 279-292, which is incorporatedherein by reference. Additional guidance in selecting and using polymersin pharmaceutical formulations can be found in texts known in the art,for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymersas Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences,Vol. 45, M. Dekker, N.Y., which is also incorporated herein byreference. Alternatively, or additionally, the virus, plasmid, or viralvector can be microencapsulated to improve administration and efficacy.Methods for microencapsulating antigens are well-known in the art, andinclude techniques described, e.g., in U.S. Pat. Nos. 3,137,631;3,959,457; 4,205,060; 4,606,940; 4,744,933; 5,132,117; and InternationalPatent Publication WO 95/28227, all of which are incorporated herein byreference.

Liposomes can also be used to provide for the sustained release ofvirus, plasmid, viral protein, or viral vector. Details concerning howto make and use liposomal formulations can be found in, among otherplaces, U.S. Pat. Nos. 4,016,100; 4,452,747; 4,921,706; 4,927,637;4,944,948; 5,008,050; and 5,009,956, all of which are incorporatedherein by reference.

An effective amount of any of the above-described vaccines can bedetermined by conventional means, starting with a low dose of virus,viral protein plasmid or viral vector, and then increasing the dosagewhile monitoring the effects. An effective amount may be obtained aftera single administration of a vaccine or after multiple administrationsof a vaccine. Known factors can be taken into consideration whendetermining an optimal dose per animal. These include the species, size,age and general condition of the animal, the presence of other drugs inthe animal, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies.

One method of detecting whether an adequate immune response has beenachieved is to determine seroconversion and antibody titer in the animalafter vaccination. The timing of vaccination and the number of boosters,if any, will preferably be determined by a doctor or veterinarian basedon analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, protein, infectious nucleotidemolecule, plasmid, or viral vector, of the present invention can bedetermined using known techniques, taking into account factors that canbe determined by one of ordinary skill in the art such as the weight ofthe animal to be vaccinated. The dose amount of virus of the presentinvention in a vaccine of the present invention preferably ranges fromabout 10¹ to about 10⁹ pfu (plaque forming units), more preferably fromabout 10² to about 10⁸ pfu, and most preferably from about 10³ to about107 pfu. The dose amount of a plasmid of the present invention in avaccine of the present invention preferably ranges from about 0.1 μg toabout 100 mg, more preferably from about 1 μg to about 10 mg, even morepreferably from about 10 μg to about 1 mg. The dose amount of aninfectious DNA molecule of the present invention in a vaccine of thepresent invention preferably ranges from about 0.1 μg to about 100 mg,more preferably from about 1 μg to about 10 mg, even more preferablyfrom about 10 μg to about 1 mg. The dose amount of a viral vector of thepresent invention in a vaccine of the present invention preferablyranges from about 10¹ pfu to about 10⁹ pfu, more preferably from about10² pfu to about 10⁸ pfu, and even more preferably from about 10³ toabout 10⁷ pfu. A suitable dosage size ranges from about 0.5 ml to about10 ml, and more preferably from about 1 ml to about 5 ml.

Suitable doses for viral protein or peptide vaccines according to thepractice of the present invention range generally from 1 to 50micrograms per dose, or higher amounts as may be determined by standardmethods, with the amount of adjuvant to be determined by recognizedmethods in regard of each such substance. In a preferred example of theinvention relating to vaccination of swine, an optimum age target forthe animals is between about 1 and 21 days, which at pre-weening, mayalso correspond with other scheduled vaccinations such as againstMycoplasma hyopneumoniae. Additionally, a preferred schedule ofvaccination for breeding sows would include similar doses, with anannual revaccination schedule.

Dosing

A preferred clinical indication is for treatment, control and preventionin both breeding sows and gilts pre-farrowing, followed by vaccinationof piglets. In a representative example (applicable to both sows andgilts), two 2-ML doses of vaccine will be used, although of course,actual volume of the dose is a function of how the vaccine isformulated, with actual dosing amounts ranging from 0.1 to 5 ML, takingalso into account the size of the animals. Single dose vaccination isalso appropriate.

The first dose may be administered as early as pre-breeding to 5-weekspre-farrowing, with the second dose administered preferably at about 1-3weeks pre-farrowing. Doses vaccine preferably provide an amount of viralmaterial that corresponds to a TCID₅₀ (tissue culture infective dose) ofbetween about 10⁶ and 10⁸, more preferably between about 10⁷ and10^(7.5), and can be further varied, as is recognized in the art.Booster doses can be given two to four weeks prior to any subsequentfarrowings. Intramuscular vaccination (all doses) is preferred, althoughone or more of the doses could be given subcutaneously. Oraladministration is also preferred. Vaccination may also be effective innaïve animals, and non-naïve animals as accomplished by planned ornatural infections.

In a further preferred example, the sow or gilt is vaccinatedintramuscularly or orally at 5-weeks pre-farrowing and then 2-weekspre-farrowing. Under these conditions, a protective immune response canbe demonstrated in PEDV-negative vaccinated sows in that they developedantibodies (measured via fluorescent focal neutralization titer fromserum samples) with neutralizing activity, and these antibodies werepassively transferred to their piglets. The protocols of the inventionare also applicable to the treatment of already seropositive sows andgilts, and also piglets and boars. Booster vaccinations can also begiven and these may be via a different route of administration. Althoughit is preferred to re-vaccinate a mother sow prior to any subsequentfarrowings, the vaccine compositions of the invention nonetheless canstill provide protection to piglets via ongoing passive transfer ofantibodies, even if the mother sow was only vaccinated in associationwith a previous farrowing.

It should be noted that piglets may then be vaccinated as early as Day 1of life. For example, piglets can be vaccinated at Day 1, with orwithout a booster dose at 3 weeks of age, particularly if the parentsow, although vaccinated pre-breeding, was not vaccinated pre-farrowing.Piglet vaccination may also be effective if the parent sow waspreviously not naïve either due to natural or planned infection.Vaccination of piglets when the mother has neither been previouslyexposed to the virus, nor vaccinated pre-farrowing may also effective.Boars (typically kept for breeding purposes) should be vaccinated onceevery 6 months. Variation of the dose amounts is well within thepractice of the art. It should be noted that the vaccines of the presentinvention are safe for use in pregnant animals (all trimesters) andneonatal swine. The vaccines of the invention are attenuated to a levelof safety (i.e. no mortality, only transient mild clinical signs orsigns normal to neonatal swine) that is acceptable for even the mostsensitive animals again including neonatal pigs. Of course, from astandpoint of protecting swine herds both from PEDV epidemics andpersistent low level PEDV occurrence, programs of sustained sowvaccination are of great importance. It will be appreciated that sows orgilts immunized with PEDV MLV will passively transfer immunity topiglets, including PEDV-specific IgA, which will protect piglets fromPEDV associated disease and mortality. Additionally, generally, pigsthat are immunized with PEDV MLV will have a decrease in amount and/orduration or be protected from shedding PEDV in their feces, and further,pigs that are immunized with PEDV MLV will be protected from weight lossand failure to gain weight due to PEDV, and further, PEDV MLV will aidin stopping or controlling the PEDV transmission cycle.

It should also be noted that animals vaccinated with the vaccines of theinvention are also immediately safe for human consumption, without anysignificant slaughter withhold, such as 21 days or less.

When provided therapeutically, the vaccine is provided in an effectiveamount upon the detection of a sign of actual infection. Suitable doseamounts for treatment of an existing infection include between about 10and about 10⁶ log₁₀ TCID₅₀, or higher, of virus per dose (minimumimmunizing dose to vaccine release). A composition is said to be“pharmacologically acceptable” if its administration can be tolerated bya recipient. Such a composition is said to be administered in a“therapeutically or prophylactically effective amount” if the amountadministered is physiologically significant.

At least one vaccine or immunogenic composition of the present inventioncan be administered by any means that achieve the intended purpose,using a pharmaceutical composition as described herein. For example,route of administration of such a composition can be by parenteral,oral, oronasal, intranasal, intratracheal, topical, subcutaneous,intramuscular, transcutaneous, intradermal, intraperitoneal,intraocular, and intravenous administration. In one embodiment of thepresent invention, the composition is administered by intramuscularly.Parenteral administration can be by bolus injection or by gradualperfusion over time. Any suitable device may be used to administer thecompositions, including syringes, droppers, needleless injectiondevices, patches, and the like. The route and device selected for usewill depend on the composition of the adjuvant, the antigen, and thesubject, and such are well known to the skilled artisan. Administrationthat is oral, or alternatively, subcutaneous, is preferred. Oraladministration may be direct, via water, or via feed (solid or liquidfeed). When provided in liquid form, the vaccine may be lyophilized withreconstitution, pr provided as a paste, for direct addition to feed (mixin or top dress) or otherwise added to water or liquid feed.

Generation of Vero Cells Suitable for Large Scale Virus Production

Viruses of the invention can be conveniently grown in Vero cell stocksthat are approved for vaccine production. To generate safe and approvedcell stock, a vial of Vero cells was subject to additional passaging.The cells were passed four times in PMEM w/wheat to produce Master CellStock (MCS) Lot “1834430”. The MCS was tested in accordance with 9CFR &EP requirements in PGM-Biological Quality Control; Lincoln, Nebr. TheMCS tested satisfactory for sterility, freedom from mycoplasmas, andextraneous agents. Therefore, PF-Vero MCS lot “1834430”, is deemedeligible for submission to the Center for Veterinary BiologicsLaboratories (CVB-L) for confirmatory testing.

Seed Origin and Passage History is as follows. A Pre-master Cell stockof global Vero cells was previously frozen. For production of the cellstock, the cells were grown in PMEM (Lincoln item #00-0779-00)containing 1% bovine serum (item #00-0710-00, BSE compliant) and 3 mML-glutamine. They were derived from Vero WCS Pass #136, Lot #071700MCS+3, 28 Jul. 2000. The new Pre-master cell stock was frozen at pass#166, which is MCS+33 from the original global Vero master cell stock.MCS “1833440” was produced from a pre-Master identified as Vero KZOpreMaster, Lot All cultures were grown in PMEM w/wheat, 1.0% L-glutamineand 1.0% Bovine Calf serum. Cells were planted (passage #167) in 150 cm2T-Flasks on Aug. 14, 2008. The flasks were incubated in 5.0% CO2 at 36 1C for 7 days then expanded. (passage #168) After flasks reached 100%confluency 4 days later, the cultures were passed (#169) into 850 cm2roller bottles. Rollers were incubated at 36 1 C at 0.125-0.250 rpmwithout CO2. The final passage of rollers (#170) was done 4 days later.Cryopreservation was completed by adding 10.0% bovine calf serum and10.0% dimethyl sulfoxide (DMSO) to the condensed cell suspension on 2Sep. 2008. Vials were labeled as passage level #170. A total of 231containers containing 4.2 ml were placed into a controlled rate freezerthen transferred into liquid nitrogen tank for long term storage atvapor phase. The MCS was produced without the use of antibiotics. Allreagents used in MCS production were sourced from Pfizer GlobalManufacturing used for licensed antigen production in domestic andglobal markets. The MCS was produced by Pfizer's Master Seed Facility,Lincoln, Nebr.

Sterility Testing was as follows. The Master Cell Stock was tested asper 9CFR (026-ST0) and EP 2.6.1 from 29 Sep. 2008 to 13 Oct. 2008. TheMCS was found to be free of bacteria and fungal contamination.

Mycoplasma Testing and Extraneous Testing were accomplished as follows.The MCS was tested as per 9CFR (028-PU0) and EP 2.6.7. The MCS was foundto be free of any Mycoplasma contamination. Extraneous testing wascompleted as per 9CFR 113.52 using NL-BT-2 (Bovine), Vero, NL-ED-5(Equine), NL-ST-1 (Porcine), NL-DK (Canine), NL-FK (Feline) cells. TheMCS was negative for MGG, CPE and HAd and tested negative by FA for BVD,BRSV, BPV, BAV-1, BAV-5, Rabies, Reo, BTV, ERV, Equine arteritis, PPV,TGE, PAV, HEV, CD, CPV, FPL and FTP. The MCS was tested by ELISA for FIVand was found to be satisfactory.

EP extraneous testing was as per 5.2.4 (52-2002). Extraneous testingusing Bovine NL-BT-2 and EBK (Primary), Vero, NL-ED-5 (Equine), NL-ST-1(Porcine), MARC MA 104, NL-DK (Canine) NL-FK (Feline) cells werenegative for MGG, CPE, HAd and tested negative by FA for BVD, BPV,BAV-1, BAV-5, Bovine corona, Bovine rotavirus, BHV-3, PI3, IBR, BRSV andBEV-1, Reo, BTV, ERV, Equine arteritis, PPV, PRV, TGE, HEV, PAV, P. rotaA1, rota A2, PRRSV, CD, CPI, CAV-2, Measles, C. rota, Rabies, CCV, FP,FCV, FVR, FIP and FeLV.

Polypeptides and Polynucleotides of the Invention

Representative embodiments of the invention include an isolatedpolynucleotide sequence that comprises a polynucleotide selected fromthe group consisting of: (a) (SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37,39, and/or 59-77); or a fragment thereof than encodes, ORF1a/1bpolyproteins, the PEDV spike protein preferably domain 1, ORF3 protein,envelope protein, membrane protein, nucleocapsid protein or afragment(s) of said proteins; (b) the complement of any sequence in (a);(c) a polynucleotide that hybridizes with a sequence of (a) or (b) understringent conditions defined as hybridizing to filter bound DNA in 0.5MNaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C.; (d) a polynucleotide that is at least 70% identical to thepolynucleotide of (a) or (b); (e) a polynucleotide that is at least 80%identical to the polynucleotide of (a) or (b); (f) a polynucleotide thatis at least 90% identical to the polynucleotide of (a) or (b); and (g) apolynucleotide that is at least 95% identical to the polynucleotide of(a) or (b). In a preferred embodiment the polynucleotide includes asecond heterologous polynucleotide sequence.

The invention also provides a polypeptide encoded by any of the openreading frames of the genotype of SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36,37, 39, 59-78, combinations thereof, or a polypeptide that is at least90% identical thereto, domains thereof, or to a fragment thereof,including the option that additional otherwise identical amino acids arereplaced by conservative substitutions.

The invention also provides a polypeptide encoded by any of the openreading frames of the variant PEDV strain of the invention, preferablythe spike protein, or more preferably spike protein S1 domain, or apolypeptide that is at least 90% identical thereto, or to a fragmentthereof, including the option that additional otherwise identical aminoacids are replaced by conservative substitutions.

In a preferred embodiment, the polypeptide is expressed from the first1170 nucleotides of the S1 region of spike protein of the variant PEDVstrain of the invention.

In a further preferred embodiment, there are further provided PEDVpolypeptide-based vaccines wherein the antigen is defined by: a proteinencoded by an open reading frames of SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36,37, 39, 59-77, combinations thereof, or an immunogenic fragment thereof.Further embodiments include an amino acid sequence where the antigen is:encoded by the nucleotides of the SEQ ID NOs: 23-34, 40-58.

Further Genetic Manipulations

The polynucleotide and amino acid sequence information provided by thepresent invention also makes possible the systematic analysis of thestructure and function of the viral genes and their encoded geneproducts. Knowledge of a polynucleotide encoding a viral gene product ofthe invention also makes available anti-sense polynucleotides whichrecognize and hybridize to polynucleotides encoding a polypeptide of theinvention, or a fragment thereof. Full length and fragment anti-sensepolynucleotides are useful in this respect. The worker of ordinary skillwill appreciate that fragment anti-sense molecules of the inventioninclude (i) those which specifically recognize and hybridize to aspecific RNA (as determined by sequence comparison of DNA encoding aviral polypeptide of the invention as well as (ii) those which recognizeand hybridize to RNA encoding variants of the encoded proteins.Antisense polynucleotides that hybridize to RNA/DNA encoding other PEDVpeptides are also identifiable through sequence comparison to identifycharacteristic, or signature sequences for the family of molecules,further of use in the study of antigenic domains in PEDV polypeptides,and may also be used to distinguish between infection of a host animalwith remotely related non-PEDV members of the Circoviridae.

Guidance for effective codon optimization for enhanced expression inyeast and E. coli for the constructs of the invention is generally knownto those of skill in the art.

Antibodies

Also contemplated by the present invention are anti-PEDV antibodies(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, humanized, human, porcine, and CDR-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a PEDV polypeptide of the invention. The term“specific for” indicates that the variable regions of the antibodies ofthe invention recognize and bind a PEDV polypeptide exclusively (i.e.,are able to distinguish a single PEDV polypeptide from relatedpolypeptides despite sequence identity, homology, or similarity found inthe family of polypeptides), and which are permitted (optionally) tointeract with other proteins (for example, S. aureus protein A or otherantibodies in ELISA techniques) through interactions with sequencesoutside the variable region of the antibodies, and in particular, in theconstant region of the Ab molecule. Screening assays to determinebinding specificity of an antibody of the invention are well known androutinely practiced in the art. For a comprehensive discussion of suchassays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of the PEDV polypeptides ofthe invention are also contemplated, provided that the antibodies arefirst and foremost specific for, as defined above, a PEDV polypeptide ofthe invention from which the fragment was derived.

For the purposes of clarity, “antibody” refers to an immunoglobulinmolecule that can bind to a specific antigen as the result of an immuneresponse to that antigen. Immunoglobulins are serum proteins composed of“light” and “heavy” polypeptide chains having “constant” and “variable”regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM)based on the composition of the constant regions. Antibodies can existin a variety of forms including, for example, as, Fv, Fab′, F(ab′) 2, aswell as in single chains, and include synthetic polypeptides thatcontain all or part of one or more antibody single chain polypeptidesequences.

Diagnostic Kits

The present invention also provides diagnostic kits. The kit can bevaluable for differentiating between porcine animals naturally infectedwith a field strain of a PEDV virus and porcine animals vaccinated withany of the PEDV vaccines described herein. The kits can also be of valuebecause animals potentially infected with field strains of PEDV viruscan be detected prior to the existence of clinical symptoms and removedfrom the herd, or kept in isolation away from naive or vaccinatedanimals. The kits include reagents for analyzing a sample from a porcineanimal for the presence of antibodies to a particular component of aspecified PEDV virus. Diagnostic kits of the present invention caninclude as a component a peptide or peptides from the variant PEDVstrain of the invention which is present in a field strain but not in avaccine of interest, or vice versa, and selection of such suitablepeptide domains is made possible by the extensive amino acid sequencing.As is known in the art, kits of the present invention can alternativelyinclude as a component a peptide which is provided via a fusion protein.The term “fusion peptide” or “fusion protein” for purposes of thepresent invention means a single polypeptide chain consisting of atleast a portion of a PEDV virus protein, preferably of ORF1, or ORF3 anda heterologous peptide or protein.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

All the inventions disclosed herein are made under a Joint ResearchAgreement (as defined by 35 USC 100(h), 37 CFR 1.9(e)) that existsbetween the inventors' respective assignees, Iowa State University andZoetis Services LLC, and the inventions are thus eligible for theexamination protections accorded by 37 CFR 1.104 (C)(4)(5).

EXAMPLES Example 1

PEDV S1 (first 2.2 kb portion of the spike gene) sequencing wasundertaken to help determine the genetic relatedness and molecularepidemiology of PEDV in US swine. Sequencing was performed on 15 PEDVcases at ISU VDL in January 2014. Among them, PEDV S1 sequences from 10cases (ISU cases 6-15) are similar to each other and to the PEDV strainsidentified in US swine in 2013 (99.1-100% nucleotide identities). Indistinct contrast, the PEDV S1 sequences from the other 5 cases (ISUcases 1-5) only have 93.9-94.6% nucleotide identities to the PEDVstrains previously identified in US swine in 2013.

However, these 5 PEDV cases shared 99.6-100% nucleotide identities toeach other based on the S1 sequences. Phylogenetic analysis based on the51 sequences demonstrated that the aforementioned 10 PEDV cases (ISUcases 6-15) clustered together with PEDV strains identified in US sinceApril 2013. However, the aforementioned 5 PEDV cases (ISU cases 1-5)clustered very differently from the PEDV strains previously identifiedin US swine (FIG. 1). Sequence alignment showed that the S1 sequences ofthese 5 PEDV cases had some deletions and insertions compared to PEDVviruses previously identified in US.

The S1 gene of this new virus isolate has been determined and isreported herein as SEQ ID NO: 1. Based on the data currently available,it appears unlikely that this strain is a mutant evolved from PEDVpreviously identified in US swine. The PEDV real-time RT-PCR offered atISU VDL is targeting the nucleocapsid (N) gene. The N-gene is known tobe a conserved portion of the PEDV genome. Thus far, the PEDV N-genereal-time RT-PCR being conducted at the ISU VDL seems to be readilydetecting these new PEDVs. The full-length N gene sequences of the newPEDVs have been determined and were similar to the PEDVs previouslyidentified in the US.

FIG. 2 shows phylogenetic trees based on the S1 portion sequences andthe whole genome sequences. See the attachment. One can see that, inaddition to in US swine, the US prototype-like strains have beendetected in South Korea, Canada and Mexico; the US variant-INDEL-likestrains have been detected in South Korea, Mexico and Germany.

Example 2

At least two genetically different porcine epidemic diarrhea virus(PEDV) strains have been identified in the United States: U.S. PEDVprototype and S-INDEL-variant strains. The objective of this study wasto compare the pathogenicity differences of the U.S. PEDV prototype andS-INDEL-variant strains in conventional neonatal piglets underexperimental infections. Fifty PEDV-negative 5-day-old pigs were dividedinto 5 groups of 10 pigs each and were inoculated orogastrically withthree U.S. PEDV prototype isolates (IN19338/2013-P7, NC35140/2013-P7,and NC49469/2013-P7), an S-INDEL-variant isolate (2014020697-P7), andvirus-negative culture medium, respectively, with virus titers of 10⁴TCID50/ml, 10 ml per pig. All three PEDV prototype isolates tested inthis study, regardless of their phylogenetic clades, had similarpathogenicity and caused severe enteric disease in 5-day-old pigs asevidenced by clinical signs, fecal virus shedding, and gross andhistopathological lesions. Compared to pigs inoculated with the threeU.S. PEDV prototype isolates, pigs inoculated with the S-INDEL-variantisolate had significantly diminished clinical signs, virus shedding infeces, gross lesions in small intestines, ceca and colons,histopathological lesions in small intestines and immunohistochemistrystaining in ileum. The U.S. PEDV prototype and the S-INDEL-variantstrains induced similar viremia levels in inoculated pigs. Whole genomesequences of the PEDV prototype and S-INDEL-variant strains weredetermined but the molecular basis of virulence differences betweenthese PEDV strains remains to be elucidated using reverse geneticsapproach. The present study provides a strong foundation to theunderstanding of the molecular mechanisms which contribute to PEDVvirulence and vaccine attenuation.

Results

Isolation and Sequence Comparison of U.S. PEDVs

Three U.S. PEDV prototype isolates USA/NC35140/2013, USA/IA49379/2013and USA/NC49469/2013 were isolated in Vero cells. Typical PEDVcytopathic effects including syncytial body formation and celldetachment were observed and virus growth was confirmed byimmunofluorescence staining. All isolates grew efficiently in Vero cellsand the infectious titers ranged from 103-106 TCID50/ml for the firstten passages.

The whole genome sequences of the three U.S. PEDV prototype isolatesUSA/NC35140/2013-P7, USA/IA49379/2013-P7, and USA/NC49469/2013-P7 weredetermined and compared to those of the previously described U.S. PEDVprototype isolate USA/IN19338/2013 and U.S. PEDV S-INDEL-variant isolateUSA/IL20697/2014 with results summarized in Table 3. Schematic diagramsof PEDV genome organization and putative functions of viral proteins aredescribed in FIG. 12. The prototype isolates USA/IN19338/2013-P7,USA/NC35140/2013-P7, USA/IA49379/2013-P7 and USA/NC49469/2013-P7 all hada genome of 28,038 nucleotides in length and had 99.75-99.91% nucleotide(nt) identity (26-69 nt differences) to each other at the whole genomelevel. The spike genes of these prototype isolates all had 4,161nucleotides in length and had 99.54-99.88% nt identity (5-19 ntdifferences) to each other. The S-INDEL-variant isolate 2014020697-P7had a genome of 28,029 nucleotides in length and had 99.08-99.22% ntidentity (220-259 nt differences) at the whole genome level to the fourprototype isolates evaluated in this study. Among them, about 64-96 ntdifferences were located in ORF1a/1b region especially the nsp12 andnsp16 regions; however, a majority of these nt changes on nsp12 andnsp16 were synonymous (silent) changes at the amino acid level (Table3). Striking differences between the U.S. PEDV prototype andS-INDEL-variant isolates were located in the spike gene (96.25-96.37% ntidentity; 151-156 nt differences) especially the S1 portion(93.14-93.32% nt identity; 148-152 nt differences); the nucleotidechanges at the S1 portion resulted in changes of the deduced amino acids(Table 3). Compared to the prototype isolates, the S gene of the variantisolate 2014020697-P7 had three characteristic deletions (a 1-ntdeletion of G at position 167, an 11-nt deletion of AGGGTGTCAAT atpositions 176-186, and a 3-nt deletion of ATA at positions 416-418) andone insertion (a 6-nt insertion of CAGGAT between positions 474 and475).

Phylogenetic analyses of the PEDV isolates described in this study and45 PEDV reference sequences are provided in FIG. 6. In the whole genomesequence-based neighbor-joining tree FIG. 6A, the U.S. PEDVprototype-like strains clustered together, which can be further dividedinto clade 1 and clade 2; however, the U.S. PEDV S-INDEL-variant-likestrains clustered separately. In the whole genome sequence-based maximumlikelihood tree FIG. 6B, the U.S. PEDV prototype-like strains alsoclustered into clade 1 and clade 2; however, the S-INDEL-variant-likestrains formed a separate sublineage within clade 2. In contrast, thephylogenetic clusters in the S1 sequence-based neighbor-joining treeFIG. 6C and the maximum likelihood tree FIG. 6D were similar. In bothFIG. 6C and FIG. 6D, the U.S. PEDV prototype-like strains clusteredtogether which can be further divided into clade 1 and clade 2, similarto the whole genome sequence-based neighbor-joining tree FIG. 6A; theU.S. PEDV S-INDEL-variant-like strains formed a separate branch that wasmore closely related to some classical PEDV isolates such asEurope/CV777, South Korea/SM98, and China/SD-M which have the samepattern of insertions and deletions in the S gene as the U.S. PEDVS-INDEL-variant-like strains.

The prototype isolates IN19338 and IA49379 belong to the clade 1 and theisolate NC35140 belongs to the clade 2 regardless of the wholegenome-based trees FIG. 6A, 6B or the S1-based trees FIG. 6C, 6D.However, the prototype isolate NC49469 belongs to the clade 1 in thewhole genome-based trees FIG. 6A, 6B but belongs to the clade 2 in theS1-based trees FIG. 6C, 6D. Three prototype isolates USA/IN19338/2013-P7(SEQ ID NO:59), USA/NC35140/2013-P7 (SEQ ID NO:60), andUSA/NC49469/2013-P7 (SEQ ID NO:61) and one S-INDEL-variant isolate2014020697-P7 (SEQ ID NO:62) were selected to compare their pathogenesisin pigs (Table 4).

TABLE 3 Nucleotide and amino acid differences between the U.S. PEDVprototype and S-INDEL-variant isolates Nucleotide and amino aciddifferences between the Nucleotide and amino Prototype isolates aciddifferences among (IN19338, IA49379, the Prototype isolates NC49469, andNC35140) (IN19338, IA49379, and the S-INDEL-variant NC49469, andNC35140) isolate IL20697 Genome region or ORF Protein(s) and region(s)No. of nt No. of aa No. of nt diff No. of aa (nucleotide position)*(length in amino acids) diff (%)¹ diff (%)¹ (%) diff (%) Whole genome(1-28038) 26-69 (0.09-0.25%) 220-259 (0.78-0.92%) 5′ UTR (1-292) N/A^(‡)0-2 (0-0.68%) N/A 0-2 (0-0.68%) N/A Nonstructural proteins (nsp) ORF1ab(293-20637) 1ab polyprotein (6781) 16-50 (0.08-0.25%) 9-16 (0.13-0.23%)64-96 (0.31-0.47%) 7-19 (0.10-0.28%) nsp1: Met1-Gly110 0 (0%) 0 (0%) 0(0%) 0 (0%) (110) nsp2: Asn111-Gly895 5-13 (0.21-0.55%) 3-5 (0.38-0.64%)4-17 (0.17-0.72%) 1-6 (0.13-0.76%) (785) nsp3: Gly896-Gly2516 5-22(0.10-0.45%) 3-9 (0.18-0.56%) 6-24 (0.12-0.49%) 1-10 (0.06-0.62%) 1621)nsp4: Ala2517-Gln2997 0-1 (0-0.07%) 0 (0%) 1-2 (0.07-0.14%) 0 (0%) (481)nsp5: Ala2998-Gln3299 0-2 (0-0.22%) 0 (0%) 0-1 (0-0.11%) 0 (0%) (302)nsp6: Ser3300-Gln3579 0-2 (0-0.24%) 0 (0%) 0-1 (0-0.12%) 0 (0%) (280)nsp7: Ser3580-Gln3662 0 (0%) 0 (0%) 1 (0.40%) 0 (0%) (83) nsp8:Ser3663-Gln3857 0 (0%) 0 (0%) 0 (0%) 0 (0%) (195) nsp9: Asn3858-Gln39650 (0%) 0 (0%) 0 (0%) 0 (0%) (108) nsp10: Ala3966-Gln4100 0-1 (0-0.25%) 0(0%) 0-1 (0-0.25%) 0 (0%) (135) nsp12: Ser4101-Gln5027 0-6 (0-0.22%) 0-1(0-0.11%) 23-27 (0.83-0.97%) 0-1 (0-0.11%) (927) nsp13: Ser5028-Gln55461-2 (0.06-0.12%) 0 (0%) 2-3 (0.12-0.19%) 0 (0%) (519) nsp14:Asn5547-Gln6141 0-3 (0-0.17%) 0-1 (0-0.17%) 1-2 (0.06-0.11%) 0-1(0-0.17%) (595) nsp15: Gly6142-Gln6480 0-2 (0-0.20%) 0-2 (0-0.59%) 8-9(0.79-0.88%) 1-2 (0.29-0.59%) (339) nsp16: Ala6481-Lys6781 0-1 (0-0.11%)0-1 (0-0.33%) 12-13 (1.32-1.43%) 1-2 (0.33-0.66%) (301) Structural andaccessory proteins Spike (20634-24794) S (1386) 5-19 (0.12-0.46%) 3-10(0.22-0.72%) 151-156 (3.63-3.75%) 59-65 (4.26-4.69%) Spike 1(20634-22847) S1 (738) 4-13 (0.18-0.59%) 3-7 (0.41-0.95%) 148-152(6.68-6.86%) 58-61 (7.86-8.27%) Spike 2 (22848-24794) S2 (648) 0-6(0-0.31%) 0-3 (0-0.46%) 3-8 (0.15-0.41%) 1-4 (0.15-0.62%) ORF3(24794-25468) NS3B (224) 0-3 (0-0.44%) 0-1 (0-0.45%) 1-3 (0.15-0.44%)0-1 (0-0.45%) Envelope (25449-25679) E (76) 0-1 (0-0.43%) 0-1 (0-1.31%)0-1 (0-0.43%) 0-1 (0-1.31%) Membrane (25687-26367) M (226) 0 (0%) 0 (0%)0 (0%) 0 (0%) Nucleocapsid (26379-27704) N (441) 1-2 (0.07-0.15%) 0-2(0-0.45%) 3-4 (0.23-0.30%) 1-2 (0.23-0.45%) 3′ UTR (27705-28038) N/A 0(0%) N/A 0 (0%) N/A *Nucleotides are numbered according to the sequenceof the U.S. PEDV prototype isolate USA/IN19338/2013 (GenBank accessionnumber KF650371). ¹Percentage of nucleotide (nt) and amino acid (aa)differences were calculated at each gene or protein level. ^(‡)N/A: Notapplicable.

TABLE 4 Experimental design of 5-day-old pigs inoculated with variousPEDV isolates Necropsy Necropsy Group Piglets U.S. PEDV Strain Inoculum(3 DPI) (7 DPI) G1 N = 10 Prototype isolate 10⁴ TCID₅₀/ml; N = 5 N = 5USA/IN19338/2013-P7 10 ml G2 N = 10 Prototype isolate 10⁴ TCID₅₀/ml; N =5 N = 5 USA/NC35140/2013-P7 10 ml G3 N = 10 Prototype isolate 10⁴TCID₅₀/ml; N = 5 N = 5 USA/NC49469/2013-P7 10 ml G4 N = 10S-INDEL-variant isolate 10⁴ TCID₅₀/ml; N = 5 N = 5 USA/IL20697/2014-P710 ml G5 N = 10 Virus-negative culture 10 ml N = 5 N = 5 mediumClinical Assessment

All pigs in G1 (USA/IN19338/2013-P7), G2 (USA/NC35140/2013-P7), and G3(USA/NC49469/2013-P7) developed soft to watery diarrhea starting from 1DPI and continuing through 6 or 7 DPI. In contrast, in G4 (IL20697),only 1 pig had mild diarrhea with soft feces at 1 DPI. The averagediarrhea scores are summarized in FIG. 7A. Overall, pigs in G1 (P=0.001)and G3 (P<0.0001) had significantly higher average diarrhea scores thanpigs in G2 when 0-7 DPI diarrhea scores were analyzed as described inthe ‘Materials and Methods’ section below. Pigs in G1-G3 inoculated withthe prototype PEDV isolates overall had significantly higher averagediarrhea scores than G4 (P<0.0001) inoculated with the PEDV variantisolate and G5 (negative control, P<0.0001). The average diarrhea scoreswere not significantly different between G4 and G5 (P=1).

No vomiting was observed from any pigs throughout the study. In G1-G3inoculated with the prototype isolates: 1) almost all pigs lost theirappetite during the study period and tube feeding had to beadministered; 2) severe dehydration, rough hair, flat or thin flankswere observed in all pigs with most severe body conditions at about 4DPI; 3) various degrees of lethargy including head down and recumbencewere observed from 1 DPI to the end of the study. In contrast, in G4inoculated with the variant isolate: 1) all pigs had normal appetite; 2)no dehydration or lethargy was observed; 3) 90% pigs had mild flatflanks at 1 DPI or 2 DPI but recovered to normal after 4 DPI. All G5pigs were active without diarrhea, dehydration, lethargy or anorexiaduring the study period.

From (−1) to 3 DPI, PEDV-inoculated pigs (G1-G4) had significantly loweraverage daily gain (ADG, P≤0.0001) compared to pigs in G5 (negativecontrol), but there were no significant differences in ADG (P-valuesranged from 0.089-1) among G1-G4 (FIG. 7B). From (−1) to 7 DPI, G1-G3(prototype isolates) had significantly lower ADG (P-values ranged from0.01-0.037) than G4 (variant isolate) although none of the G1, G2, G3,or G4 had significant difference in ADG (P-values ranged from0.078-0.847) compared to G5 (negative control) (FIG. 7B).

Virus Shedding and Distribution

PEDV RNA was detected in rectal swab samples from all pigs in G1-G3(prototype isolates) at 1 DPI until the end of the study. In G4 (variantisolate), PEDV RNA was detected in rectal swabs from 5/10, 8/10, 10/10,5/5, 5/5, 3/5, and 2/5 pigs at 1, 2, 3, 4, 5, 6 and 7 DPI, respectively.The average genomic copies per ml of virus shed in rectal swabs aresummarized in FIG. 7C. Pigs in G1 and G2 had similar levels (P=0.601) offecal virus shedding from 1-7 DPI with the quantity ranging from107.2-9.0 genomic copies/ml, corresponding to Ct values 16-22. Pigs inG3 had the highest level of fecal virus shedding at 1-2 DPI(approximately 109 genomic copies/ml with Ct 16) and the fecal virusshedding gradually declined to approximately 105.4 genomic copies/ml at7 DPI, corresponding to Ct value 28. In contrast, pigs in G4 had about102.3 genomic copies/ml (Ct 31.8) fecal virus shedding at 1 DPI; thefecal virus shedding gradually increased and peaked at 5 DPI (105.4genomic copies/ml with Ct 28.8) and then declined to approximately 101.3genomic copies/ml (Ct 36.3) at 7 DPI. Statistical analyses indicatedthat G1-G3 (prototype isolates) had significantly larger amounts ofviral RNA shedding in rectal swabs (P<0.0001) compared to G4 (variantisolate).

PEDV RNA was detected in serum samples from all pigs in G1-G4 necropsiedat 3 DPI with average Ct values of 33.2 (G1), 30.5 (G2), 31.9 (G3) and28.4 (G4). There were no significant differences between average PEDVgenomic copies in sera of G1-G4 at 3 DPI [P-values ranged from0.077-0.646, FIG. 7D. At 7 DPI, PEDV RNA was detected in serum samplesfrom 3-4 out of 5 pigs in G1-G4 with average Ct values (only onPCR-positive pigs) of 33.5 (G1), 34.2 (G2), 37.4 (G3) and 35.8 (G4). Theaverage genomic copies of PEDV in sera of G4 (variant isolate) had nosignificant difference (P-values ranged from 0.050-0.717) compared tothose in G1-G3 (prototype isolates) at 7 DPI FIG. 7D.

Virus distributions in tissues are summarized in Table 5. At 3 DPI,regardless of G1, G2, G3 or G4, average PEDV RNA concentrations inileums, ceca, colons and mesenteric lymph nodes were higher than theconcentrations in other tissues within the same inoculation group. Whenviral RNA concentrations in each tissue type were compared across fourinoculation groups at 3 DPI, viral RNA concentrations in cecum and colonof G4 (variant isolate) were overall significantly lower than in cecumand colon of G1-G3 (prototype isolates); however, viral RNAconcentrations in other tissues of G4 were similar to those in thecorresponding tissues of G1-G3; the same types of tissues in G1-G3 hadsimilar levels of viral RNA. Data at 7 DPI overall supported similarconclusions to 3 DPI except that viral genomic copies in cecum and colonwere much lower than the genomic copies in ileum and mesenteric lymphnode in G4.

All rectal swabs, sera, and tissue samples from G5 (negative control)were negative by PEDV real-time RT-PCR throughout the study period.

TABLE 5 PCR detection of PEDV RNA in various tissues at 3 DPI and 7 DPIfrom pigs inoculated with four PEDVs. IL20697 (Variant; G4) IN19338(Prototype; G1) NC35140 (Prototype; G2) NC49469 (Prototype; G3) PCR PCRPCR PCR Spec- posi- Mean Genomic posi- Mean Genomic posi- Mean Genomicposi- Mean Genomic imen tive Ct⁺ copies/ml^(‡) tive Ct⁺ copies/ml^(‡)tive Ct⁺ copies/ml^(‡) tive Ct⁺ copies/ml^(‡) 3 Stomach 5/5 31.9 3.25 ×10⁴A 5/5 23.9 7.45 × 10⁶A 4/5 25.9 1.07 × 10⁵A 5/5 27.9 5.06 × 10⁵A DPIIleum 5/5 17.1 7.49 × 10⁸B 5/5 16.5   1.17 × 10⁹AB 5/5 16.4   1.22 ×10⁹AB 5/5 15.8 1.88 × 10⁹A Cecum 5/5 22.5 2.01 × 10⁷C 5/5 20.5   7.81 ×10⁷BC 5/5 19.9 1.12 × 10⁸B 5/5 16.9 8.55 × 10⁸A Colon 5/5 25.0 3.58 ×10⁶B 5/5 15.7 1.90 × 10⁹A 5/5 16.8 9.44 × 10⁸A 5/5 14.3 5.02 × 10⁹ATonsil 4/5 32.4 3.19 × 10³A 5/5 30.1 1.13 × 10⁵A 5/5 32.7 1.98 × 10⁴A4/5 32.7 2.63 × 10³A Heart 5/5 34.6 5.33 × 10³A 4/5 35.7 5.28 × 10²A 4/535.4 6.30 × 10²A 4/5 36.4 3.53 × 10²A Lung 3/5 34.2 2.07 × 10²A 2/5 36.71.76 × 10¹A 1/5 36.4 4.36 × 10⁰A 2/5 36.0 2.10 × 10¹A Liver 5/5 32.03.14 × 10⁴A 4/5 33.4 1.83 × 10³A 3/5 33.3 2.91 × 10²A 4/5 34.2 1.16 ×10³A Spleen 5/5 32.1 2.93 × 10⁴A 5/5 33.8 9.44 × 10³A 4/5 33.3 1.89 ×10³A 5/5 34.9 4.37 × 10³A Kidney 3/5 34.4 1.84 × 10²A 3/5 34.3 1.99 ×10²A 3/5 36.9 6.70 × 10¹A 1/5 35.9 4.65 × 10⁰A MLN 5/5 21.3   4.52 ×10⁷AB 5/5 19.3 1.76 × 10⁸A 5/5 22.9 1.46 × 10⁷B 5/5 21.1   5.21 × 10⁷ABMuscle* 5/5 33.9 8.77 × 10³B 5/5 30.8 7.26 × 10⁴A 5/5 33.5   1.09 ×10⁴AB 5/5 34.9 4.26 × 10³B 7 Stomach 0/5 >45 0B 3/5 36.1 9.57 × 10¹A 3/532.1   3.11 × 10¹AB 1/5 36.7   4.16 × 10⁰AB DPI Ileum 5/5 25.8 2.15 ×10⁶B 5/5 24.0   7.29 × 10⁶AB 5/5 21.6 3.54 × 10⁷A 5/5 24.4   5.36 ×10⁶AB Cecum 5/5 36.0 2.12 × 10³B 5/5 28.9 2.57 × 10⁵A 5/5 26.4 1.38 ×10⁶A 4/5 29.1   1.92 × 10⁴AB Colon 1/5 34.2 5.85 × 10⁰B 5/5 23.9   7.37× 10⁶AB 5/5 22.1 2.58 × 10⁷A 4/5 26.6 7.23 × 10⁴A Tonsil 4/5 36.2 3.99 ×10²A 5/5 32.6 2.13 × 10⁴A 4/5 35.5 2.99 × 10³A 3/5 34.5 1.79 × 10²AHeart 0/5 >45 0A 2/5 36.1 2.08 × 10¹A 3/5 36.5 1.14 × 10¹A 0/5 >45 0ALung 0/5 >45 0A 1/5 34.6 5.58 × 10⁰A 3/5 32.0 3.17 × 10¹A 0/5 >45 0ALiver 0/5 >45 0A 1/5 32.4 7.46 × 10⁰A 2/5 37.5 5.24 × 10⁰A 0/5 >45 0ASpleen 2/5 36.5 1.87 × 10¹A 3/5 35.8 1.04 × 10²A 3/5 34.0 2.02 × 10¹A1/5 38.2 3.43 × 10⁰A Kidney 0/5 >45 0A 1/5 38.1 3.47 × 10⁰A 3/5 35.41.47 × 10¹A 0/5 >45 0A MLN 5/5 26.2 1.57 × 10⁶A 5/5 26.3 1.50 × 10⁶A 5/524.9 3.74 × 10⁶A 5/5 27.3 7.63 × 10⁵A Muscle* 0/5 >45 0B 3/5 35.5   1.20× 10²AB 3/5 30.9 2.54 × 10²A 0/5 >45 0B Abbreviations: DPI, days postinoculation; MLN, mesenteric lymph node; PCR polymerase chain reaction;Ct, cycle threshold. *Rear leg muscle. ⁺Mean Ct was mean Ct value ofPCR-positive pigs (pigs with PCR Ct < 45). ^(‡)Genomic copies/ml wasgeometric mean of genomic copies per milliter of all pigs (bothPCR-positive and negative pigs). Staistical analyses were performed onthe same tissue types for G1-G4 groups and different letters indicatesignificant differences.Gross Pathology

At 3 DPI, thin and transparent walls, sometimes dilated by gas and/oryellowish fluid, were observed in small intestine, cecum and colontissues in most pigs inoculated with the PEDV prototype isolates(G1-G3). In addition, almost all pigs in G1-G3 contained watery contentsin small intestines, ceca and colons. In contrast, only mildly thinwalls could be observed in small intestines of 3/5 pigs inoculated withthe PEDV variant isolate (G4); no apparent gross lesions were observedin ceca or colons of pigs in G4. Also, in G4, only 1/5 pigs had waterycontents in small intestine, cecum and colon; two other pigs hadsemi-watery contents in cecum. Statistically, all G1-G3 (prototypeisolates) had significantly higher small intestine, cecum and coloncontent scores (P-values ranged from <0.0001 to 0.0127) than G4 (variantisolate) and G5 (negative control) FIG. 8A. A significant difference wasnot observed among G1-G3 (P-values ranged from 0.3722-1) for smallintestine, cecum, and colon content scores FIG. 8A. For G4, only cecumcontent scores were significantly higher than G5 (P=0.003), but notsmall intestine or colon content scores [P-values ranged from 0.5259-1;FIG. 8A]. All prototype isolate-inoculated groups (G1-G3) hadsignificantly higher tissue lesion scores on small intestine, cecum, andcolon (P-values ranged from 0.0012-0.049) than the variantisolate-inoculated group (G4) and the negative control group (G5) FIG.8B. No significant differences were observed on small intestine, cecumand colon lesion scores among G1-G3 (P-values ranged from 0.1585-1) orbetween G4 and G5 [P-values ranged from 0.4766-1; FIG. 8B].

At 7 DPI, most pigs in G1-G3 still had thin-walled and/or gas-distendedsmall intestines but only about 50% of pigs in G1-G3 had thin-walledand/or gas-distended ceca and colons. All pigs in G1 and G2 and 4/5 pigsin G3 had watery small intestine contents. About 60-100% of pigs inG1-G3 had semi-watery or watery contents in ceca and colons. In G4, only1/5 pig had thin-walled small intestines and none of the pigs had grosslesions in ceca and colons. In G4, 5/5, 1/5 and 0/5 pigs had semi-wateryor watery contents in small intestines, ceca, and colons, respectively.In G5, 1/5 pigs had thin-walled small intestine, cecum and colon; 3/5pigs had semi-watery or watery contents in small intestines, ceca andcolons. Significant differences on tissue content scores and tissuelesion scores were observed between some groups (FIG. 8).

Rectal swabs collected at 3 DPI and 7 DPI were confirmed negative forPDCoV, TGEV, and porcine rotaviruses (groups A, B &C) and negative forhemolytic E. coli and Salmonella spp.

Histopathology

No remarkable microscopic lesions were observed in stomach, cecum,colon, tonsil, mesenteric lymph node, heart, lung, liver, spleen, andkidney sections of all piglets (G1-G5) at either 3 DPI or 7 DPI.

Severe lesions consistent with viral enteritis (e.g. villous enterocyteswelling, villous atrophy, collapsed lamina propria etc.) were observedin small intestinal sections (duodenum, jejunum, and ileum) of all pigsin G1-G3 (prototype isolates) necropsied at 3 DPI and 7 DPI. Mildmicroscopic lesions consistent with viral enteritis were observed insmall intestinal sections of pigs in G4 (variant isolate) necropsied at3 DPI; however, microscopic lesions were not remarkable in smallintestinal sections of pigs in G4 necropsied at 7 DPI. Microscopiclesions were not apparent in small intestinal sections of pigs in G5(negative control) at either 3 DPI or 7 DPI. Representative images ofH&E-stained ileum sections of pigs necropsied at 3 DPI from G1-G5 areshown in FIG. 9A-9E.

Villus height, crypt depth, and villus-height-to-crypt-depth ratio weremeasured and compared on small intestinal sections of 5 inoculationgroups. At 3 DPI, pigs in G1-G3 (prototype isolates) had significantlydecreased average villus heights, increased average crypt depths, andlower average villus/crypt ratios in duodenum, proximal jejunum, middlejejunum, distal jejunum, and ileum than pigs in G4 (variant isolate) andG5 (negative control) with some exceptions (FIG. 10); exceptions includevillus height and villus/crypt ratio in duodenum of G2 and G4 as well ascrypt depth in middle jejunum of G1 and G4. The average villus heights,crypt depths, and villus/crypt ratios of small intestine sections at 3DPI were overall similar across the three groups G1-G3 inoculated withthe prototype isolates (FIG. 10). The average crypt depths of all smallintestinal sections at 3 DPI were not significantly different between G4(variant isolate) and G5 (negative control); however, pigs in G4 hadsignificantly decreased average villus heights and lower averagevillus/crypt ratios in duodenum, middle and distal jejunum and ileum at3 DPI compared to pigs in G5 (FIG. 10).

At 7 DPI, the average villus heights and villus/crypt ratios of smallintestinal sections were overall similar across the three groups G1-G3FIG. 11A-11D. Pigs in G1-G3 overall had significantly decreased averagevillus heights and lower average villus/crypt ratios in small intestinalsections than pigs in G4 and G5 at 7 DPI (FIG. 11A, 11C). Interestingly,the average villus heights at 7 DPI were either not significantlydifferent between G4 (variant isolate) and G5 (negative control) or weresignificantly higher in G4 than G5 (FIG. 11A). The average villus/cryptratios at 7 DPI were not significantly different in proximal, middle anddistal jejunum, and ileum between G4 and G5 although the averagevillus/crypt ratios in duodenum were significantly different between G4and G5 (FIG. 10C). Comparison of average crypt depths at 7 DPI ispresented in FIG. 11B. The average crypt depths were similar in allsmall intestinal sections between the prototype isolate-inoculatedgroups G2 and G3; both G2 and G3 had significantly longer crypt depthscompared to the negative control group G5. Another prototype-isolateinoculated group G1 had the average crypt depth values that were betweenthe negative control group G5 and the prototype isolate-inoculatedgroups G2 and G3. The variant isolate-inoculated group G4 hadsignificantly increased average crypt depths in duodenum, proximal anddistal jejunums compared to the negative control group G5, while G4 hadsimilar average crypt depths to G1, G2, and G3 in most of the smallintestinal sections.

Immunohistochemistry (IHC)

At 3 DPI, PEDV-specific IHC staining was performed on serial sections ofileum, cecum and colon of all 5 inoculation groups. None of the 5 pigsin G5 (negative control) were IHC positive in the ileum, cecum or colon.All 5 pigs in each of G1-G4 were IHC positive in the ileum with averageIHC scores of 3.9 (G1), 3.7 (G2), 3.8 (G3) and 2.5 (G4). The average IHCscores for ileum were similar across G1-G3 but were significantly higherthan G4 (FIG. 10D). Regarding IHC staining of ceca, 5/5 (G1), 4/5 (G2),5/5 (G3), and 3/5 (G4) pigs were positive, with no significantdifferences on average IHC scores among G1-G4 (FIG. 10D). For colons,5/5 (G1), 4/5 (G2), 4/5 (G3) and 2/5 (G4) pigs were IHC stainingpositive but the average IHC scores were not significantly differentamong G1-G4 (FIG. 10D). Representative PEDV IHC staining images areshown in FIG. 9F-9T.

At 7 DPI, PEDV IHC staining was only performed on serial sections ofileum. Mild/scant IHC staining was observed in G1 and G2 but no stainingwas observed in G3, G4 and G5 (FIG. 11D).

DISCUSSION

Sequence analyses demonstrated that at least two genetically differentPEDV strains have been circulating in the U.S. (Vlasova et al., 2014;Wang et al., 2014), and they are referred as U.S. PEDV prototype strainand U.S. PEDV S-INDEL-variant strain. The U.S. prototype PEDVs can bephylogenetically further divided into clade 1 and clade 2. In the wholegenome sequence-based phylogenetic analyses, the U.S.S-INDEL-variant-like PEDVs clustered separately from the clade 1 andclade 2 in the neighbor-joining tree but they formed a separatesublineage within clade 2 in the maximum likelihood tree (FIG. 6A, 6B).This suggests that phylogenetic analysis tools and tree constructionmethods could result in some differences in the outcomes of theanalyses; thus, conclusions should be drawn cautiously by clearlyindicating the tools and methods used for phylogenetic analyses. AmongU.S. prototype PEDVs, some always belong to clade 1 or clade 2regardless of whole genome-based trees or S1-based trees; however, some(e.g. NC49469 and Minnesota62) belong to clade 1 in whole genome-basedtrees but belong to clade 2 in S1-based trees (FIG. 6). It is probablybecause the S1 sequences of NC49469 and Minnesota62 PEDVs are moreclosely related to clade 2 but the remaining genome sequences are moreclosely related to clade 1 PEDVs. Our group has isolated various PEDVsin cell culture that fall into each category described above, enablingus to compare the pathogenesis of various U.S. prototype and5-INDEL-variant PEDVs.

Studies have demonstrated that neonatal piglets are more susceptiblethan weaned pigs to PEDV infection and PEDV infection induces greaterdisease severity in neonates than in weaned pigs (Jung et al., 2015a;Thomas et al., 2015). Therefore, a sensitive 5-day-old neonatal pigletmodel was selected for pathogenesis comparisons in this study. Amongthree U.S. PEDV prototype isolates (USA/IN19338/2013, USA/NC35140/2013,and USA/NC49469/2013), the average diarrhea scores induced by theUSA/NC35140/2013-P7 isolate were lower than those by theUSA/IN19338/2013-P7 and USA/NC49469/2013-P7 isolates; however, threeprototype isolates had similar virus shedding, gross lesions,histopathological lesions, and IHC staining. Overall, we conclude thatthree U.S. PEDV prototype isolates evaluated in this study have similarpathogenicity in neonatal piglets regardless of their phylogeneticclades. In contrast, data in the current study clearly demonstrate thatthe U.S. PEDV S-INDEL-variant isolate 2014020697-P7 had significantlydiminished clinical signs, virus shedding in feces, gross lesions insmall intestines, ceca and colons, histopathological lesions in smallintestines, and IHC scores in ileum, compared to three U.S. PEDVprototype isolates USA/IN19338/2013-P7, USA/NC35140/2013-P7 andUSA/NC49469/2013-P7. Recent experimental studies by other groups alsodemonstrated that S-INDEL PEDVs overall had lower pathogenicity comparedto the U.S. prototype strains in 3-4 day old pigs or 1-week-old pigs(Lin et al., 2015a; Yamamoto et al., 2015). However, in Lin et al study,they observed that three litters of piglets inoculated with a U.S.S-INDEL Iowa106 strain had zero mortality but one litter of pigletsinoculated with the same virus strain had 75% mortality (Lin et al.,2015a). They hypothesize that the sows' health condition can have adirect impact on colostrum/milk production and thus affect the infectionoutcome of their piglets (Lin et al., 2015a). The virulence of S-INDELPEDVs observed in the field has variations among farms and countries. Inthe U.S., the S-INDEL variant strain OH851 infection only caused minimalto no clinical signs in suckling piglets on the farm (Wang et al.,2014). In Germany, two sow farms were infected with an S-INDEL PEDV thathas 99.4% nucleotide identity to the U.S. S-INDEL variant OH851 at thewhole genome level; however, severity of clinical signs and mortality insuckling piglets varied significantly between the two farms (Stadler etal., 2015). Factors contributing to the contradictory findings have notbeen clearly identified. But source of viruses (wild type orcell-culture adapted viruses), inoculation/infection doses,animal/environmental conditions, and nucleotide/amino acid variationsamong S-INDEL PEDVs could contribute to the observed discrepancies amongvarious experimental studies and field outbreaks. In addition, PEDVpathogenicity can be age-dependent. Further investigations ofpathogenicity of S-INDEL PEDV variants in weaned pigs, finisher pigs,gilts and sows are warranted.

Studies have shown that viremia can occur in the acute stage ofinfection with U.S. PEDV prototype isolates (Jung et al., 2014; Madsonet al., 2016). In the present study, we also detected PEDV RNA in serumsamples. In addition, the PEDV variant isolate and three prototypeisolates had similar viremia levels under the conditions of this study.PEDV is an enteropathogenic coronavirus that infects the villousenterocytes, resulting in villous atrophy and malabsorptive diarrhea.Some quantities of PEDV could be taken into the blood stream throughmechanisms not-fully-understood. But PEDV is not believed to activelyreplicate in blood and viremia levels may not necessarily correlate tovirulence/pathogenicity. In the current study, high levels of PEDV RNAwere detected in small intestine, cecum, colon and mesenteric lymphnodes while low levels of PEDV RNA were detected in non-enteric tissues(tonsil, heart, lung, liver, spleen, kidney, and muscle) from pigsinoculated with either prototype or S-INDEL-variant PEDV. Previousstudies indicated that PEDV viral antigen (U.S. prototype isolates)could be detected in small intestine, mesenteric lymph node, and somecolon and spleen tissues (Jung et al., 2015b; Jung et al., 2014; Madsonet al., 2016) but other non-enteric tissues such as lung, heart, kidneyand liver were all negative for PEDV antigen (Madson et al., 2016).Therefore, detection of PEDV RNA does not necessarily mean that PEDVreplicates in all of these non-enteric tissues. Considering that theblood was not drained before collecting each organ, the possibility thatvirus in these tissues was from blood cannot be excluded.

In the current study, PEDV IHC staining was only performed on ileum,cecum and colon of inoculated pigs. Among four groups inoculated withPEDVs (three prototype isolates and one variant isolate), PEDV IHCstaining was observed in 100% ileums, 60-100% ceca, and 40-100% colonsat 3 DPI. The average IHC scores in ileums were significantly lower inthe variant isolate-inoculated pigs than in the prototypeisolates-inoculated pigs, consistent with observations on grosspathology and histopathological lesions of small intestines. Althoughthe average IHC scores in ceca and colons were numerically lower in pigsinoculated with the variant isolate than in pigs inoculated with threeprototype isolates, the differences were not significant. However, PEDVvariant isolate-inoculated pigs had fewer gross changes in cecum andcolon than the prototype isolate-inoculated pigs. Thus, it may need tobe further elucidated about the correlations of cecal and colonicchanges to PEDV virulence/pathogenicity.

All four groups G1-G4 inoculated with PEDVs (three prototype isolatesand one variant isolate) had significantly shortened villus heightscompared to the negative control group G5 at 3 DPI and G1-G3 (prototypeisolates) had significantly shortened villus heights compared to G4(variant isolate). These indicate that both U.S. prototype and variantPEDV isolates can infect and destroy villus epithelium of smallintestines, but the U.S. PEDV variant isolate caused less severe villousatrophy than prototype isolates. Intestinal crypt epithelial cells serveto replace the destroyed villous enterocytes. At 3 DPI, the averagecrypt depths of G4 (variant isolate) were not significantly differentfrom G5 (negative control) but the average crypt depths of G1-G3(prototype isolates) were significantly longer than G4 and G5. These maysuggest that mild villous atrophy caused by U.S. PEDV variant isolatehas not triggered significant proliferation and elongation of intestinalcrypt at 3 DPI; however, intestinal crypts have started to elongate tosome degree to repair severe villous atrophy in prototypeisolate-inoculated groups G1-G3. At 7 DPI, prototype isolate-inoculatedgroups had longer average crypt depth than the negative control group,suggesting that crypt elongation continued to replace the damaged villusenterocytes but the villus epithelium has not recovered back to normal.The average crypt depths of some G4 (variant isolate) sections of smallintestine were significantly longer than G5 (negative control) at 7 DPI,suggesting that elongation of crypts occurred later in G4 compared tothe prototype isolate-inoculated groups G1-G3. The proliferated cryptseventually recovered the destroyed villus enterocytes apparent at 3 DPIin G4. IHC staining also supported these observations.

Studies have shown that the antibodies against U.S. PEDV prototype andS-INDEL-variant strains can cross-react and cross-neutralize bothstrains in vitro (Chen et al., 2016; Lin et al., 2015b). An in vivostudy (Goede et al., 2015) showed that sows exposed to S-INDEL-variantPEDV infection 7 months ago could provide partial protection to newbornpiglets challenged with a U.S. PEDV prototype strain. Another in vivostudy (Lin et al., 2015a) demonstrated that 3-4 days old piglets exposedto S-INDEL-variant PEDV could partially protect against subsequentchallenge with a U.S. prototype PEDV. Applicants also have data thatdemonstrates both U.S. PEDV prototype and S-INDEL-variant strains canprovide homologous and heterologous protection against two virus strainsin a weaned pig model. In the current study, it was demonstrated thatU.S. PEDV S-INDEL-variant strain is less virulent than U.S. PEDVprototype strains in neonatal pigs. These data collectively suggest thatU.S PEDV S-INDEL-variant strain could potentially be a modified livevirus vaccine candidate against PED although additional evaluation workis needed to determine overall efficacy (see Examples 5 and 6 foradditional work).

The striking sequence differences between U.S. prototype andS-NDEL-variant PEDVs are located in the spike gene especially the S1portion. The sequence differences in the spike gene may be partiallyresponsible for the virulence differences between U.S. prototype andS-INDEL-variant PEDVs.

Materials and Methods

Virus Isolates and Cells

Isolation and characterization of the U.S. PEDV prototype isolateUSA/IN19338/2013 and S-INDEL-variant isolate USA/IL20697/2014 have beendescribed (Chen et al., 2014; Chen et al., 2016). Three additional U.S.PEDV prototype isolates USA/NC35140/2013, USA/IA49379/2013 andUSA/NC49469/2013 were obtained for this study, all from archived pigletfeces submitted to the Iowa State University Veterinary DiagnosticLaboratory (ISU VDL) for routine diagnosis, following previouslydescribed virus isolation procedures (Chen et al., 2014). All PEDVisolation, propagation and titration were performed in Vero cells (ATCCCCL-81) as described (Chen et al., 2014). All PEDV isolates used in thisstudy were confirmed negative for porcine deltacoronavirus (PDCoV),transmissible gastroenteritis virus (TGEV), and porcine rotaviruses(groups A, B, & C), porcine reproductive and respiratory syndrome virus,and porcine circovirus.

Virus Sequencing, Comparative Sequence Analysis and PhylogeneticAnalysis

The whole genome sequences of the PEDV isolates described in this studywere determined by next generation sequencing technology using IlluminaMiSeq platform and assembled with SeqMan Pro version 11.2.1 (DNAstarInc, Madison, Wis.) as described previously (Chen et al., 2014). Thesequence data of these PEDV isolates were deposited to GenBank with thefollowing accession numbers: USA/IN19338/2013 [KF650371],USA/NC35140/2013-P7 [KM975735], USA/IA49379/2013 [KM975736],USA/NC49469/2013-P7 [KM975737], and 2014020697-P7 [KT860508].

The whole genome sequences and individual gene sequences (nucleotide andamino acid sequences) of all PEDV isolates used in this study werealigned using ClustalX version 2.0 (Larkin et al., 2007) and BioEditversion 7.0.4.1 (Hall, 1999) to compare the genetic similarity.Phylogenetic analysis was conducted using the entire genome and the S1portion (S gene nucleotides 1 to 2205 according to the sequenceKF650371) nucleotide sequences of the PEDV isolates described in thisstudy as well as representative global PEDVs (in total 50 sequences).Phylogenetic trees, respectively, were constructed using thedistance-based neighbor-joining method and maximum likelihood method ofMEGA version 6 (Tamura et al., 2013). Bootstrap analysis was carried outon 1,000 replicate dataset.

Experimental Design

The animal study protocol was approved by the Iowa State UniversityInstitutional Animal Care and Use Committee (Approval No. 6-14-7821-S;approved on 10th of July 2014). Fifty 5-day-old piglets were purchasedfrom a conventional breeding farm and delivered to the Iowa StateUniversity Laboratory Animal Resources facilities. All pigs wereintramuscularly injected with a dose of Excede® (Zoetis, Florham Park,N.J., USA) upon arrival and confirmed negative for PEDV, PDCoV, TGEV,and porcine rotaviruses (groups A, B, & C) by virus-specific PCRs onrectal swabs and negative for PEDV antibody by a virus-specific indirectfluorescent antibody (IFA) assay on serum samples at the ISU VDL. Pigswere blocked by weight and then randomly divided into 5 groups of 10pigs each, one group per room on a solid floor. Pigs were fed a mixtureof Esbilac (Hampshire, Ill.) liquid milk replacer and yogurt and hadfree access to water. After 1-day acclimation (piglets were 6 days old),pigs in groups 1 through 5 (G1-G5) were orogastrically inoculated withthree U.S. PEDV prototype isolates USA/IN19338/2013-P7 (G1),USA/NC35140/2013-P7 (G2), USA/NC49469/2013-P7 (G3), one U.S.S-INDEL-variant isolate 2014020697-P7 (G4), or virus-negative culturemedium (G5), respectively (10 ml/pig; all viruses were at the 7thpassage in cell culture with the titer of 104 TCID50/ml) (Table 4).

Piglets were evaluated daily for presence of vomiting and clinical signsof diarrhea, lethargy, and body condition. Diarrhea severity was scoredwith the following criteria: 0=normal, 1=soft (cowpie), 2=liquid withsome solid content, 3=watery with no solid content. Lethargy levels werecategorized as normal, mild lethargy (slow to move, head down), moderatelethargy (stands but wants to lie down), or severe lethargy (recumbent,moribund). Body condition was categorized as: normal, mild loss (flatflank), moderate (flank tucked in), or severe (backbone/ribs prominent).

Body weights were recorded prior to inoculation and then at 3 and 7 dayspost inoculation (DPI). The average daily gain (ADG) was calculated onpigs from (−1) to 3 DPI and (−1) to 7 DPI. Serum samples were collectedat 0, 3, and 7 DPI. Rectal swabs were collected daily from each pig from0 DPI to necropsy and were submerged into 1 ml PBS immediately aftercollection. Five pigs from each group were randomly selected fornecropsy at 3 DPI, and the remaining pigs were necropsied at 7 DPI.Fresh and formalin-fixed samples collected at necropsy included: tonsil,heart, lung, liver, spleen, kidney, skeletal muscle from rear leg,stomach, mesenteric lymph node, duodenum, proximal jejunum, middlejejunum, distal jejunum, ileum, cecum, and colon. Collection ofdifferent intestinal segments was performed as previously described(Madson et al., 2014).

At necropsy, the small intestine, cecum and colon were examined forgross lesions by veterinary pathologists blind to the treatment groups.Tissue lesions were categorized as normal, thin-walled, and/orgas-distended. The presence of thin-walled intestines or gas-distendedorgans was numerated as 1 point, respectively; the presence of boththin-walled and gas-distended was numerated as 2 points. Contents ofsmall intestine, cecum and colon were examined and scored with thecriteria: 0=Normal, 1=liquid with some solids (semi-watery), 2=watery.

To rule out the possibility of concurrent infections with otherpathogens, rectal swabs collected at 3 DPI and 7 DPI before necropsywere tested for PDCoV, TGEV, and porcine rotaviruses (groups A, B &C) byvirus-specific PCRs and for hemolytic E. coli and Salmonella spp. byroutine bacterial cultures at ISU VDL.

Virus Shedding as Examined by a Quantitative PEDV N Gene-Based Real-TimeRT-PCR

Viral RNA was extracted from rectal swabs, serum, and 10% tissuehomogenates as previously described (Chen et al., 2014). Five μl of eachRNA template was used in PCR setup in a 25 μl total reaction usingPath-ID™ Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific). Theprimers, probes and in vitro transcribed RNA used to generate standardcurves of a quantitative PEDV N gene-based real-time RT-PCR had beendescribed (Lowe et al., 2014; Madson et al., 2014; Thomas et al., 2015).Based on standard curves, virus concentration in the unit of genomiccopies/ml in tested samples was calculated. The mean cycle threshold(Ct) values were calculated based on PCR positive samples, and the meanvirus concentrations were calculated based on all pigs within the group(both PCR positive and negative pigs).

Histopathology

Tonsil, heart, lung, liver, spleen, kidney, mesenteric lymph node,stomach, duodenum, proximal jejunum, middle jejunum, distal jejunum,ileum, cecum, and colon tissues were fixed in 10% formalin, embedded,sectioned, and stained with hematoxylin and eosin (H&E) and examined bya veterinary pathologist blinded to individual animal identificationsand treatment groups. Villus lengths and crypt depths were measured fromthree representative villi and crypts of duodenum, proximal jejunum,middle jejunum, distal jejunum, and ileum, using a computerized imagesystem following previously described procedures (Madson et al., 2014).Villus-height-to-crypt-depth (villus/crypt) ratio of each tissue wascalculated as the quotient of the average villus length divided by theaverage crypt depth.

Immunohistochemistry

Serial sections of ileum, cecum and colon at 3 DPI necropsy wereevaluated for PEDV antigen by immunohistochemistry (IHC) using aPEDV-specific monoclonal antibody (BioNote, Hwaseong-si, Gyeonggi-do,Korea) as previously described (Madson et al., 2014). At 7 DPI necropsy,IHC staining was only performed on serial sections of ileum. The IHCantigen detection was semi-quantitatively scored as previously described(Chen et al., 2015) with the following criteria: 0=no staining;1=approximately 1-10% enterocytes with positive staining;2=approximately 10%-25% enterocytes with positive staining;3=approximately 25%-50% enterocytes with positive staining;4=approximately 50%-100% enterocytes with positive staining.

Statistical Analyses

Generalized linear mixed (GLIMMIX) model was used for all statisticalcomparisons with Statistical Analysis System (SAS) version 9.3 (SASinstitute, Cary, N.C.). P-value<0.05 was defined as statisticallysignificant. P-values of overall fecal viral shedding level [Log10(genomic copies/ml)] were assessed among treatments from 0-7 DPI, withDPI and treatment as interacting variables, and similarly for analysisof diarrhea scores.

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Example 3

At least two genetically different porcine epidemic diarrhea virus(PEDV) strains have been identified in the United States (U.S. PEDVprototype and S-INDEL-variant strains). The current serological assaysoffered at veterinary diagnostic laboratories for detection ofPEDV-specific antibody are based on the U.S. PEDV prototype strain. Theobjectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variantstrain in cell culture; 2) generate antisera against the U.S. PEDVprototype and S-INDEL-variant strains by experimentally infecting weanedpigs; 3) determine if the various PEDV serological assays could detectantibodies against the U.S. PEDV S-INDEL-variant strain and vice versa.A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in thisstudy. Three groups of PEDV-negative, 3-week-old pigs (five pigs pergroup) were inoculated orally with a U.S. PEDV prototype isolate(previously isolated in our lab), an S-INDEL-variant isolate orvirus-negative culture medium. Serum samples collected at 0, 7, 14, 21and 28 days post inoculation were evaluated by the following PEDVserological assays: 1) indirect fluorescent antibody (IFA) assays usingthe prototype and S-INDEL-variant strains as indicator viruses; 2) virusneutralization (VN) tests against the prototype and S-INDEL-variantviruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDVprototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strainS1-based ELISA. The positive antisera against the prototype strainreacted to and neutralized both prototype and S-INDEL-variant viruses,and the positive antisera against the S-INDEL-variant strain alsoreacted to and neutralized both prototype and S-INDEL-variant viruses,as examined by IFA antibody assays and VN tests. Antibodies against thetwo PEDV strains could be detected by all three ELISAs althoughdetection rates varied to some degree.

Applicants show that the antibodies against U.S. PEDV prototype andS-INDEL-variant strains cross-reacted and cross-neutralized both strainsin vitro. The current serological assays based on U.S. PEDV prototypestrain can detect antibodies against both U.S. PEDV strains.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrheavirus (PEDV), was first recorded in England in the early 1970s and hassince spread to other European and Asian countries [1]. In NorthAmerica, PEDV was detected for the first time in the United States(U.S.) in April 2013 [2] and subsequently PEDV was reported in Canada[3] and Mexico [4]. PEDV is an enveloped, single-stranded,positive-sense RNA virus belonging to the order Nidovirale, the familyCoronaviridae, subfamily Coronavirinae, genus Alphacoronavirus [5]. ThePEDV genome is approximately 28 kb in length and includes ORF1a andORF1b encoding the replicase polyproteins and other opening readingframes (ORFs) encoding four structural proteins [spike (S), envelope(E), membrane (M), and nucleocapsid (N)] and one nonstructural proteinNS3B (encoded by ORF3) [1].

In the U.S., a highly virulent PEDV strain (U.S. PEDV prototype strain)was identified during the initial PED outbreaks [2, 6, 7]. Lately, aPEDV variant strain having insertions and deletions (INDEL) in the spikegene compared to the U.S. prototype strain was identified in U.S. swinewith mild clinical signs based on field observations [8]. This U.S. PEDVvariant strain, also known as S INDEL strain [4], formed a distinctphylogenetic cluster compared to U.S. PEDV prototype strains [4, 8, 9].One PEDV isolate (PC177) having a 197-aa deletion in the N-terminal Sprotein was discovered during PEDV isolation in cell culture; however,this PEDV isolate still phylogenetically clustered with the U.S. PEDVprototype strains and was not considered as one of the S-INDEL-variantstrains [10]. Marthaler et al [11] reported a ‘third’ strain of PEDV(Minnesota188) in U.S. swine that had 6 nucleotide deletions (2 aminoacid deletions) in the spike gene (different from the U.S.S-INDEL-variant strains). However, the PEDV Minnesota188 was geneticallyvery closely related to the U.S. PEDV prototype strains and it isarguable whether it should be called a ‘third’ strain of PEDV in U.S.The PEDV PC177 and Minnesota188 are probably the mutants of the U.S.PEDV prototype strains. Therefore, there are at least two geneticallydifferent PEDV strains currently circulating in U.S. swine: U.S. PEDVprototype strain and S-INDEL-variant strain.

The U.S. PEDV prototype strains have been successfully isolated andpropagated in cell culture by several groups [7, 10, 12, 13]. A numberof serological assays, including an indirect fluorescent antibody (IFA)assay, a virus neutralization (VN) test, a whole virus-basedenzyme-linked immunosorbent assay (ELISA), a recombinant S1protein-based ELISA, and recombinant nucleocapsid protein-based ELISAs,have been developed for the detection of PEDV-specific antibodies[14-18]. All of these serological assays are based on the U.S. PEDVprototype strains.

In this study, Applicants isolate a U.S. PEDV S-INDEL-variant strain incell culture. Pigs were experimentally inoculated with a U.S. PEDVprototype strain and the newly isolated U.S. PEDV S-INDEL-variantstrain, respectively, to generate strain-specific antisera.Subsequently, the generated swine antisera were subjected to an in vitroevaluation for serological cross-reactivity and cross-neutralizationbetween the two strains. Specifically, 1) PEDV IFA antibody assays(using the prototype and S-INDEL-variant strains as indicator viruses,respectively) and ELISAs (PEDV prototype strain whole virus-based ELISA,PEDV prototype strain S1-based ELISA, and PEDV S-INDEL-variant strainS1-based ELISA) were conducted to evaluate the antibody cross-reactivityof the two U.S. strains; and 2) VN tests using the prototype andS-INDEL-variant strains as indicator viruses were conducted to evaluatethe in vitro cross-neutralization of two U.S. strains.

Materials and Methods

Isolation of U.S. PEDV S-INDEL-Variant Strain in Cell Culture.

Sixty-eight clinical samples (27 fecal swabs, 24 feces, 13 smallintestines and 4 oral fluids), which were tested positive by a PEDV Ngene-based real-time RT-PCR [17, 19] at the Iowa State UniversityVeterinary Diagnostic Laboratory (ISU VDL) and confirmed positive forthe U.S. PEDV S-INDEL-variant strain but negative for the U.S. prototypestrain by S1 sequencing, were selected to attempt virus isolation inVero cells (ATCC CCL-81) following previously described procedures [7].

Among the aforementioned 68 clinical samples positive for the U.S. PEDVS-INDEL-variant strain, one small intestine homogenate (with PEDV Ngene-based real-time RT-PCR cycle threshold (Ct) value of 16.1) [17, 19]from a pig located in Illinois was inoculated orogastrically into threePEDV-naïve weaned pigs at 3 weeks of age (10 ml per pig). The homogenateused for inoculation was confirmed negative for transmissiblegastrointestinal virus (TGEV), porcine rotavirus groups A, B, C andporcine deltacoronavirus (PDCoV) by virus-specific RT-PCRs at the ISUVDL. Rectal swabs and feces were collected from each inoculated pigtwice a day and tested by the PEDV real-time RT-PCR on the same day.Once the RT-PCR Ct values of the rectal swabs were <15, the pig waseuthanized and necropsied within 24 hours. Small intestine tissues andcecum contents were collected for attempting virus isolation in cellculture as previously described [7]. This animal study was performedaccording to the procedures approved by the Iowa State UniversityInstitutional Animal Care and Use Committee (IACUC, approval number3-14-7766-S).

The whole genome sequence of the U.S. PEDV S-INDEL-variant strain cellculture isolate USA/IL20697/2014 obtained in this study was determinedby next-generation sequencing (NGS) technology using an Illumina MiSeqplatform as described previously [7]. The PEDV S1 portion sequences ofthe isolate USA/IL20697/2014 and the clinical sample from which thevirus isolate was derived were determined by Sanger sequencing followingthe previously described procedures [7].

Generation of Antisera Against the U.S. Prototype and S-INDEL-VariantPEDVs.

Fifteen 3-week-old pigs, negative for PEDV as confirmed by a real-timeRT-PCR on rectal swabs and by IFA antibody assay on sera, were randomlydivided by weight into 3 groups with 5 pigs per group and with similaraverage weight per group. After acclimation for 3 days, three groups ofpigs were orogastrically inoculated with a U.S. PEDV prototype cellculture isolate USA/IN19338/2013-P7 (Pro group) (SEQ ID NO:59) [7], aU.S. PEDV S-INDEL-variant cell culture isolate USA/IL20697/2014-P7 (SEQID NO:62) (Var group), and virus-negative culture medium (Neg group),respectively, with virus titers of 104 TCID50/ml, 10 ml per pig. Rectalswabs were collected from all pigs daily between 0 and 7 DPI, and thenat 10, 14, 21 and 28 DPI, and tested by a PEDV N gene-based quantitativereal-time RT-PCR [20] to confirm infection. Serum samples were collectedfrom all pigs at 0, 7, 14, 21 and 28 days post inoculation (DPI) forcross-reactivity and cross-neutralization evaluations. This animal studywas performed according to the procedures approved by the Iowa StateUniversity IACUC committee (approval number 6-14-7809-S).

Twenty-five serum samples collected at 0, 7, 14, 21, and 28 DPI from thePro group (Pro antisera), 25 serum samples collected from the Var group(Var antisera), and 25 serum samples collected from the Neg group (Negantisera), were tested by various serological assays in this study. Inaddition, one pig antiserum against the European PEDV CV777 strain, onepig antiserum against the TGEV Purdue strain, one pig antiserum againstthe porcine heamagglutinating encephalomyelitis virus (PHEV), one pigantiserum against the porcine respiratory coronavirus (PRCV), and onepig antiserum against PDCoV were included in this study for evaluations.Antisera against PEDV CV777, TGEV Purdue, and PHEV strains werepurchased from National Veterinary Service Laboratory, Ames, IowaAntisera against PRCV and PDCoV were positive control sera obtained fromthe ISU VDL.

Indirect Fluorescent Antibody (IFA) Assay.

Eighty serum samples were tested by the PEDV prototype strain-based IFA(Pro IFA) and S-INDEL-variant strain-based IFA (Var IFA) following thepreviously described procedures [20]. The PEDV prototype isolateUSA/IN19338/2013 was used as the indicator virus in the Pro IFA assayand the S-INDEL-variant isolate USA/IL20697/2014 was used as theindicator virus in the Var IFA assay. A positive signal at a serumdilution of 1:40 or higher was considered to be IFA antibody positive.

PEDV ELISAs for Antibody Detection.

The U.S. PEDV prototype strain whole virus-based indirect ELISA (ProWVELISA) was developed and validated at the ISU VDL for detection ofPEDV-specific IgG antibody [15, 16]. All serum samples were tested bythis ProWV ELISA following the procedures that had been described indetail [20]. The sample-to-positive (S/P) ratio of >0.8 was consideredantibody positive, an S/P ratio between 0.6-0.8 was considered suspect,and an S/P ratio <0.6 was considered negative.

A published U.S. PEDV prototype strain S1-based indirect ELISA (ProS1ELISA) was used to test all the serum samples in this study followingthe previously described procedures [14]. The S/P ratio of >0.2 wasconsidered antibody positive, 0.14-0.2 was considered suspect, and anS/P ratio <0.14 was considered negative.

A U.S. PEDV S-INDEL-variant strain S1-based indirect ELISA (VarS1 ELISA)was developed in this study. The region encoding the S1 portion (aa1-735) of the U.S. PEDV S-INDEL-variant strain was codon optimized andsynthesized with the addition of a 5′ Kozac sequence, a 5′ eukaryoticsignal sequence, and a 3′ 6×-His tag by GeneArt® Gene Synthesis (ThermoFisher Scientific, Waltham, Mass., USA). The resultant 2,358 base pairDNA fragment was cloned into a Zoetis proprietary eukaryotic expressionvector (pZOE15). The authenticity and orientation of the insert in therecombinant plasmid was confirmed by sequencing. The recombinant plasmidwas transiently transfected into human embryonic kidney (HEK) 293 cellsusing a Zoetis proprietary PEI transfection method. At 7 dayspost-transfection, culture supernatants were harvested and filtersterilized. The recombinant protein was purified via Ni-NTA PurificationSystem (Thermo Fisher Scientific). The optimum antigen concentration andthe optimum serum dilutions for the VarS1 ELISA were determined using acheckerboard titration. Polystyrene 96-well microtitration plates(Nunc®, Thermo Fisher Scientific) were coated (100 μl per well) withPEDV variant 51 protein and incubated overnight at 4° C. After 5 washeswith PBS, the plates were blocked (300 μl/well) with PBS containing 1%bovine serum albumin (Jackson ImmunoResearch Inc., West Grove, Pa., USA)for 2 h at 25° C. Plates were dried at 37° C. for 4 h and stored at 4°C. in a sealed bag with desiccant packs until use. Serum samples werediluted 1:50 and added to the coated plates (100 μl/well). Plates wereincubated at 25° C. for 1 h and then washed 5 times with PBS.Subsequently 100 μl of peroxidase-conjugated goat anti-porcine IgG (H+L)(Jackson ImmunoResearch Inc., West Grove, Pa., USA) at 1:25,000 dilutionwas added and plates were incubated at 25° C. for 1 h. After a washingstep, 100 μl tetramethylbenzidine-hydrogen peroxide substrate (TMB, DakoNorth America Inc., Carpinteria, Calif., USA) was added. Plates wereincubated at room temperature for 5 min and the reaction was stopped byadding 50 μl stop solution (1 M sulfuric acid). Reactions were measuredas optical density (OD) at 450 nm using an ELISA plate reader operatedwith commercial software (Biotek® Instruments Inc., Winooski, Vt., USA).The serum antibody response was presented as sample-to-positive (S/P)ratios calculated as: S/P ratio=(sample OD−negative control meanOD)/(positive control mean OD−negative control mean OD). The PEDV VarS1ELISA was validated using 29 field serum samples collected from a farmwith documented exposure to the U.S. PEDV S-INDEL-variant strain (serumsamples were collected from 29 weaned pigs one month after they werefound positive for S-INDEL-variant strain by PCR) and 20 PEDV-negativefield serum samples. The S/P ratio of >0.3 was considered antibodypositive, 0.2-0.3 was suspect, and <0.2 was negative.

Virus Neutralization (VN) Test.

Serum samples were tested by a U.S. PEDV prototype strain-based VN (ProVN) and a U.S. PEDV S-INDEL-variant strain-based VN (Var VN) followingthe previously described procedures [20]. The PEDV prototype isolateUSA/IN19338/2013 was used as the indicator virus in the Pro VN assay andthe S-INDEL-variant isolate USA/IL20697/2014 was used as the indicatorvirus in the Var VN assay. The reciprocal of the highest serum dilutionresulting in >90% reduction of staining as compared to the negativeserum control was defined as the VN titer of the serum sample. A VNtiter of >8 was considered positive.

Statistical Analysis.

The Log 2 (IFA titer/10) of the Pro antisera and the Var antisera testedby Pro IFA and Var IFA were analyzed in a generalized linear mixed model(GLIMMIX). Days post inoculation and antigen were used as independentvariables, and pig ID and the interaction of pig ID and antigen were setas random effects. The Log 2 (VN titer) of the Pro antisera and the Varantisera tested by Pro VN and Var VN were analyzed in a similar way. ForELISA analysis, ELISA antigen, pig ID and DPI were used as independentvariables. All statistical analyses were performed with StatisticalAnalysis System (SAS) version 9.3 (SAS institute, Cary, N.C., USA), withp value <0.05 considered significantly different.

Results

Isolation of the U.S. PEDV S-INDEL-Variant Strain in Cell Culture.

Virus isolation was first attempted on 68 clinical samples received atthe ISU VDL that tested positive for the U.S. PEDV S-INDEL-variantstrain but virus isolation attempts in cell culture were unsuccessful.Subsequently a PEDV S-INDEL-variant strain-positive intestine homogenatewas used to inoculate three 3-week-old pigs. The rectal swab of one pighad a PEDV RT-PCR Ct<15 at 2 DPI and the pig was euthanized andnecropsied at 3 DPI. The rectal swabs of the other two pigs had PEDVRT-PCR Ct<15 at 3 DPI and both pigs were euthanized and necropsied at 4DPI. The small intestine tissues and cecum contents collected atnecropsy were used to attempt virus isolation in Vero cells. The U.S.PEDV S-INDEL-variant strain was successfully isolated from smallintestine homogenates and cecum contents collected from all 3 pigs.Typical PEDV cytopathic effects including syncytial body formation andcell detachment were observed and the virus growth was confirmed byimmunofluorescence staining using PEDV-specific monoclonal antibodySD6-29.

One U.S. PEDV S-INDEL-variant isolate designated as USA/IL20697/2014 wasselected for further propagation and characterization. This isolate wasserially passed in Vero cells and the infectious titers ranged from103-105 TCID50/ml for the first ten passages. The whole genome sequencesof the isolate 2014020697-P5 passage 5, lineage 1 (SEQ ID NO:8) had99.3-99.9% nucleotide identity to other U.S. PEDV S-INDEL-variantsequences available in GenBank. The S1 sequences of the USA/IL20697/2014cell culture isolate P5 had 99.8% nucleotide identity (only 4 nucleotidedifferences) to the original intestine homogenate from which the virusisolate was derived. The USA/IL20697/2014 isolate was tested at the ISUVDL and confirmed negative for TGEV, PRCV, PDCoV, porcine rotavirus A,B, C, influenza A virus, porcine reproductive and respiratory syndromevirus, and porcine circovirus 2 by virus-specific PCRs.

Generation of Antisera Against the U.S. Prototype and S-INDEL-VariantPEDVs.

The U.S. PEDV prototype isolate USA/IN19338/2013-P7 (SEQ ID NO:59) andS-INDEL-variant isolate 2014020697-P7 (SEQ ID NO:62) became establishedin all inoculated pigs as evidenced by PCR testing of the rectal swabs.In prototype group, 4/5, 5/5, 5/5, 5/5, 5/5, 5/5, and 3/5 pigs shed thevirus in rectal swabs at 2, 4, 7, 10, 14, 21, and 28 DPI, respectively,as tested by PEDV real-time RT-PCR. In S-INDEL-variant group, 3/5, 5/5,5/5, 5/5, 4/5, 3/5 and 1/5 pigs shed the virus in rectal swabs at 2, 4,7, 10, 14, 21 and 28 DPI, respectively. The rectal swabs of the negativecontrol pigs remained PEDV PCR negative throughout the study period. Intotal, 25 antisera were collected from the prototype strain-inoculatedpigs (Pro antisera), 25 antisera collected from the variantstrain-inoculated pigs (Var antisera), and 25 antisera collected fromnegative control group (Neg antisera), at 0, 7, 14, 21, and 28 DPI.

Evaluation of Cross-Reactivity of Antibodies Against the U.S. PEDVPrototype Strain and S-INDEL-Variant Strain by PEDV IFA Antibody Assays.

As shown in FIG. 13, the Pro antisera tested antibody negative (0/5) at0 and 7 DPI and 100% positive (5/5) at 14, 21, and 28 DPI by theprototype strain-based IFA antibody assay (Pro IFA). The variantstrain-based IFA (Var IFA) gave similar results on the Pro antiseraexcept that one serum collected at 14 DPI was negative by the Var IFAassay. When the antibody titers were compared, the positive Pro antiseraoverall reacted better to the Pro IFA assay than to the Var IFA assay,with 1.4 log 2 higher titer on average (FIG. 13).

The Var antisera tested negative (0/5) at 0 and 7 DPI and 100% positive(5/5) at 14, 21, and 28 DPI by both the Pro IFA and Var IFA antibodyassays. When the antibody titers were compared, the positive Varantisera reacted similarly to both Pro IFA and Var IFA assays, with lessthan 0.1 log 2 titer differences on average (FIG. 13).

The antisera collected from the negative control group (Neg antisera)were antibody negative by both PEDV Pro IFA and Var IFA assaysthroughout the study. The pig antiserum against the European PEDV CV777strain had similar antibody titers by the Pro IFA assay (titer 320) andby the Var IFA assay (titer 160). The antisera against TGEV Purdue,PHEV, PDCoV, and PRCV viruses were all negative by both PEDV Pro IFA andVar IFA assays.

Evaluation of Cross-Reactivity of Antibodies Against the U.S. PEDVPrototype Strain and S-INDEL-Variant Strain by Various PEDV ELISAs.

As shown in FIG. 14, the Pro antisera collected at 0 and 7 DPI were allantibody negative by ProWV ELISA, ProS1 ELISA, and VarS1 ELISA. For thePro antisera collected at 14 DPI, 2 sera were positive and 3 were in thesuspect range by the ProWV ELISA; 3 positives and 1 suspect by the ProS1ELISA; 2 positives and 1 suspect by the VarS1 ELISA. The Pro antiseracollected at 21 and 28 DPI were all positive by three ELISAs. Whencomparing the total number of positive Pro antisera at 14, 21 and 28 DPIby each ELISA, there were no significant differences among three ELISAsto detect antibody against the U.S. PEDV prototype strain.

The Var antisera collected at 0 and 7 DPI were antibody negative by allthree ELISAs, with the exception of one serum at 7 DPI that was in thesuspect range by the ProS1 ELISA (FIG. 14). The Var antisera collectedat 14, 21 and 28 DPI had variable numbers of positive, suspect andnegative results by three ELISAs (FIG. 14). Overall for the Varantisera, the ProWV ELISA detected 14 sera as antibody positive, 1 assuspect, and 10 as negative; the ProS1 ELISA detected 8 sera aspositive, 5 as suspect, and 12 as negative; the VarS1 ELISA detected 12sera as positive, 3 as suspect, and 10 as negative. When comparing thetotal number of positive Var antisera at 14, 21 and 28 DPI by eachELISA, the ProWV ELISA was significantly better than the ProS1 ELISA todetect antibody against the U.S. PEDV S-INDEL-variant strain (p=0.0079).However, there were no significant differences between the ProWV ELISAand VarS1 ELISA (p=0.3643), or between the ProS1 ELISA and VarS1 ELISA(p=0.0723), to detect antibody against the U.S. PEDV S-INDEL-variantstrain.

The antisera collected from the negative control group (Neg antisera)were antibody negative by all three PEDV ELISAs throughout the studyperiod 0-28 DPI. The pig antiserum against the European PEDV CV777strain was antibody positive by all three PEDV ELISAs. The antiseraagainst TGEV Purdue, PHEV, PDCoV, and PRCV viruses were all negative bythree PEDV ELISAs.

Evaluation of Cross-Neutralization of Antibodies Against the U.S. PEDVPrototype Strain and S-INDEL-Variant Strain by Virus NeutralizationTests.

As shown in FIG. 15, VN antibodies were detected as early as 7 DPI insera of most of the pigs inoculated with either a prototype strain or anS-INDEL-variant strain, regardless of testing by Pro VN or Var VNassays. Serum samples collected at 14, 21 and 28 DPI from all pigsinoculated with PEDV prototype strain or S-INDEL-variant strain were VNantibody positive by both Pro VN and Var VN assays.

The positive Pro antisera had similar VN antibody titers by the Pro VNand Var VN assays and there was no significant difference between thetwo assays. The positive Var antisera had similar VN antibody titers bythe Pro VN and Var VN assays and overall there was no significantdifference between the two assays (p=0.42) although the average VNantibody titers of Var antisera at 21 and 28 DPI were slightly higher bythe Var VN assay than by the Pro VN assay (FIG. 15).

The VN antibody titers of the positive Pro antisera tested by thehomologous Pro VN assay were, on average, 0.8 log 2 higher than the VNantibody titers of the positive Var antisera tested by the homologousVar VN assay (FIG. 15).

The antisera collected from the negative control group (Neg antisera)were antibody negative by both Pro VN and Var VN assays throughout thestudy period 0-28 DPI. The pig antiserum against the European PEDV CV777strain was antibody positive by the Pro VN assay (titer 64) and by theVar VN assay (titer 16). The antisera against TGEV Purdue, PHEV, PDCoV,and PRCV viruses were all negative by both PEDV Pro VN and Var VNassays.

DISCUSSION

Applicants previously isolated the U.S. PEDV prototype strains in Verocells [7]. In order to obtain a cell culture isolate of U.S. PEDVS-INDEL-variant strains to generate strain-specific antisera forevaluation, virus isolation was first attempted in Vero cells using 68PEDV S-INDEL-variant strain-positive clinical samples submitted to theISU VDL. However, it was unsuccessful to isolate an S-INDEL-variantvirus in cell culture from these samples. This could be due to multiplefactors such as low concentration of virus in samples, cytotoxicity ofsome samples, and variable storage conditions of the clinical samplesafter collection. Next, among the 68 clinical samples, one intestinehomogenate containing the S-INDEL-variant PEDV was inoculated into pigsto generate more fresh materials with abundant virus load for virusisolation attempts in cell culture. Using this approach, U.S.S-INDEL-variant PEDV was successfully isolated in Vero cells. It isspeculated that high concentration of virus in the samples and immediatevirus isolation attempts on the fresh samples are the key to success ofvirus isolation in cell culture. For other viruses under occasions thatthere is difficulty to isolate those viruses in cell cultures directlyfrom the clinical samples of naturally infected animals, the approachdescribed in this study can be considered, namely amplifying the virusin host animals to obtain fresh samples with high concentration of virusfor virus isolation attempts in cell cultures.

Some field serum samples collected from swine farms were submitted tothe ISU VDL for PEDV antibody detection. However, due to the lack ofclear exposure history of these cases as well as the possibility ofinfection with multiple pathogens or with more than one PEDV strain,these field serum samples were not ideal for evaluating serologicalcross-reactivity of different PEDV strains. Therefore, in the presentstudy, antisera against the U.S. PEDV prototype and S-INDEL-variantstrains were generated in weaned pigs under strict experimentalconditions, for evaluation of cross-reactivity by various serologicalassays.

The positive antisera against the prototype strain reacted with bothprototype and S-INDEL-variant viruses, and the positive antisera againstthe S-INDEL-variant strain also reacted with both prototype andS-INDEL-variant viruses, as examined by IFA antibody assays. When takingthe antibody titers into consideration, antibodies against the prototypestrain reacted better to the Pro IFA assay than to the Var IFA assaywhereas antibodies against the S-INDEL-variant strain reacted similarlyto the Var IFA and Pro IFA assays. Thus, the current U.S. PEDV prototypestrain-based WA antibody assay offered at veterinary diagnosticlaboratories can be used to reliably detect antibodies against both U.S.PEDV strains.

The ProWV ELISA and ProS1 ELISA have been previously developed andvalidated to detect PEDV-specific antibodies [14-16]. A PEDV VarS1 ELISAwas developed in this study. However, this VarS1 ELISA was onlyvalidated using a limited number of field antisera against the U.S. PEDVS-INDEL-variant strain before testing the experimentally generatedantisera in this study. Further validation of this VarS1 ELISA usinglarge number of serum samples would be needed to determine theperformance of this assay. All three PEDV ELISAs reacted with the Proantisera and Var antisera. The three ELISAs detected Pro antiserasimilarly. However, it appeared that the ProS1 ELISA used in this studywas not as efficient as the ProWV ELISA and the VarS1 ELISA to detectthe antibodies against the U.S. PEDV S-INDEL-variant strain under theconditions of this study.

The antibodies against the U.S. prototype strain and the antibodiesagainst the U.S. S-INDEL-variant strain neutralized both virus strainsto similar titers. The U.S. PEDV prototype strain-based VN testscurrently run in the laboratories can be used to detect antibodiesagainst both U.S. PEDV strains.

Both the prototype and S-INDEL-variant PEDV-inoculated pigs developeddetectable IFA and ELISA antibodies in sera starting from 14 DPI in thisstudy. In contrast, both groups of pigs developed low levels of serumneutralizing antibodies starting from 7 DPI. The IFA and ELISA assays inthis study detected IgG antibodies; the VN tests could potentiallydetect any antibody isotype with neutralizing activity. It is unclearwhether this contributes to the observed early detection of low-level VNantibody. In a previous study, it has also been reported that PEDV VNantibody could be detected as early as 7 DPI [20].

The distinct genetic differences between the U.S. prototype andS-INDEL-variant PEDVs are located in the S1 region (nucleotides 1-2214corresponding to aa 1-738, according to positions in the prototypestrain USA/IN19338/2013, GenBank accession number KF650371), especiallythe N-terminal region of the S gene (nucleotides 1-1170 corresponding toaa 1-390) whereas the remaining portions of the genomes are relativelyconserved between the two U.S. strains [4, 8, 10]. The PEDV prototypestrain S1 protein used for the ProS1 ELISA and the PEDV S-INDEL-variantstrain S1 protein used for the VarS1 ELISA had 92% amino acid identity.The reported PEDV neutralizing epitopes are located in the S proteinamino acid residues 499-638, 744-759, 756-771, and 1368-1374 [1, 21].The protein sequences in these locations harboring the neutralizingepitopes are conserved between the U.S. prototype and S-INDEL-variantPEDVs. This can explain why the antibodies against the two PEDV strainswere able to cross-neutralize two virus strains. The ProS1 and VarS1ELISAs were developed using the recombinant PEDV S1 proteins (aa 1-738).Although the U.S. prototype and S-INDEL-variant PEDVs have considerabledifferences in aa 1-390, the two strains still have some common epitopesin this region. In addition, the recombinant S1 proteins of two PEDVstrains have relatively conserved sequences from aa 390-738 includingthe neutralizing epitopes in this region. These may be the reasons whythe ProS1 and VarS1 ELISAs can detect antibodies against both U.S. PEDVstrains despite of possible differences on the sensitivity betweenassays. The IFA antibody assay and ProWV ELISA are supposed to detectantibodies against multiple antigenic proteins of PEDV and thus they areexpected to detect antibodies against both U.S. prototype andS-INDEL-variant PEDVs. Considering that the nucleocapsid protein israther conserved among PEDVs, it is expected that the nucleocapsidprotein-based ELISAs should detect antibodies against both U.S. PEDVstrains. Applicants also included one pig antiserum against theclassical European PEDV CV777 strain for evaluation and the PEDV CV777antibody was detected by all serological assays evaluated in this study.However, antisera against TGEV Purdue, PHEV, PDCoV and PRCV had nocross-reactivity with PEDV serological assays evaluated in this study.

Lin et al [22], hyperimmune pig antisera against U.S. PEDV prototypestrain, U.S. PEDV S-INDEL-variant strain, TGEV Purdue strain, and TGEVMiller strain were generated and tested by cell cultureimmunofluorescence (CCIF) assay (similar to our IFA antibody assay) andfluorescent focus reduction virus neutralization (FFRVN) assay (similarto our VN test). It was found that antisera against the U.S. PEDVprototype strain, S-INDEL-variant strain, and European CV777 strain allhad cross-reactivity by CCIF and FFRVN assays. Our findings areconsistent with their results. In addition to similar serological assaysused by Lin et al, we also evaluated PEDV serological reactivity viathree PEDV ELISAs. Also, we tested sequential serum samples (0-28 DPI)from pigs experimentally infected with two U.S. PEDV strains, providinguseful information about the kinetics of PEDV antibody production inweaned pigs. An interesting finding in the Lin et al study was thathyperimmune antisera against TGEV Miller strain rather than TGEV Purduestrain cross-reacted with all PEDV strains by CCIF assay but not byFFRVN assay.

CONCLUSIONS

The data in the present study indicate that the antibodies against U.S.PEDV prototype and S-INDEL-variant strains cross-reacted andcross-neutralized both strains in vitro. The current serological assaysbased on U.S. PEDV prototype strain can detect antibodies against bothU.S. PEDV strains. However, the cross-protection efficacy of these twoPEDV strains needs to be determined by in vivo pig studies. Goede et al[23] showed that sows exposed to S-INDEL-variant PEDV infection 7 monthsago could provide partial protection to newborn piglets challenged witha U.S. PEDV prototype strain. But more in vivo studies in this respectare needed to reveal whether a U.S. PEDV prototype strain or theS-INDEL-variant strain or both should be used to develop a vaccine forproviding protection against both PEDV strains circulating in U.S. swine(see Examples 5 and 6 for additional work to this regard).

REFERENCES

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Example 4

In response to current major epidemics of PEDV, and the lack ofeffective vaccines, the present invention provides a major achievement,the provision of modified live vaccines, based on INDEL-type variants (afurther example of which being OH851, as first described by the OhioDepartment of Agriculture, L. Wang et al., Emerg. Infect. Dis., 2014, v.20, pp. 917-919) that are effective against the current spectrum ofworldwide epidemics, and particularly are cross protective againstso-called prototype viruses (an example being well knownUSA/Colorado/2013, the sequence of which is available under GenBankAccession No. KF272920), all as generally described above, which havecaused devastating losses in swine herds. The completely novel, first inclass, vaccines of the present invention also accord substantialflexibility as to dosing, and timing of dosing, being effective foradministration, for example, to sows, both before and during pregnancy,and for administration to piglets, including to those born of naïvesows. Effectiveness as to boars is also provided. As a result, suchvaccines, in both adjuvanted and non-adjuvanted forms, provideprotection to sows, gilts, piglets, hogs and boars against challengewith PEDV, including prototype and emerging INDEL-type isolates. Thepresent vaccines will therefore provide previously unattainableprotective benefits, to include the achievement successful (1)vaccination of pregnant sows and gilts for protection, prevention, aidin prevention, or aid in control against disease caused by PEDV(anorexia, weight loss, dehydration, fever, diarrhea, vomiting, poorlactational performance, poor reproduction performance); (2) vaccinationof pregnant sows and gilts for protection, prevention, aid inprevention, or aid in control against disease and mortality caused byPEDV in piglets; (3) vaccination of swine 1 day of age or older forprotection, prevention, aid in prevention, or aid in control againstdisease caused by PEDV; (4) booster vaccinations to be administered tosows prior to subsequent farrowings or annually; (5) that healthy swinemay be vaccinated with the PEDV MLV and then boostered with PEDVinactivated vaccine; and (6) generally for the protection of swineagainst weight loss or failure to gain weight caused by PEDV, all andthe like.

Attenuation of Prototype Strain USA/IN19338/2013

Since its emergence in the U.S. in April 2013, porcine epidemic diarrheavirus (PEDV) has spread rapidly across the country and resulted in theestimated death of 8 million pigs in the first year, causing economiclosses of $900 million to $1.8 billion [1]. In addition, PEDV hasrecently emerged or re-emerged in a number of countries including China,Japan, South Korea, Philippines, Thailand, Vietnam, Canada, Mexico,Germany, Belgium, France and Portugal [2-10]. So PEDV still remains asignificant challenge to the global swine industries. Currently in U.S.,there are two commercial PEDV vaccines (a killed vaccine and a RNAparticle vaccine). However, some studies showed that these PEDV vaccinesinduced good IgG and IgA immune responses in herds previously exposed toPEDV but did not induce good IgA response in naïve pigs aftervaccination [11-12]. Modified live virus (MLV) PEDV vaccine is moreefficacious than the killed or subunit vaccines for inducing mucosalimmunity. However, such a safe and efficacious MLV PEDV vaccine againstthe emerging U.S. strains does not exist currently. The objectives ofthis study were to characterize the genomic and pathogenic changes of aU.S. virulent PEDV prototype isolate after serial passages in cellculture and determine if any resultant viruses could be potential MLVvaccine candidate against PED.

Materials and Methods

A U.S. PEDV prototype strain cell culture isolate USA/IN19338/2013,previously isolated in our laboratory [13], was serially passed in Verocells for 100 passages. Pathogenic changes of selected viruses wereevaluated in a neonatal pig model. Sixty piglets (5-day-old), free ofPED virus and antibody, were randomly divided into 6 groups with 10 pigsper group. Pigs were fed with milk replacer. Groups 1 through 5 (G1-5)were orally inoculated with PEDV USA/IN19338/2013 at the passages 7 (SEQID NO:59), 25 (SEQ ID NO:71), 50 (SEQ ID NO:72), 75 (SEQ ID NO:74) and100 (SEQ ID NO:75), respectively (10⁴ TCID50/ml, 10 ml/pig) and Group 6(G6) received the same volume of virus-free culture media as negativecontrol (Table 6). Clinical observations were recorded. Rectal swabswere collected at arrival and daily and tested by a PEDV nucleocapsidgene-based quantitative real-time RT-PCR14. Five pigs from each groupwere necropsied at 3 and 7 days post inoculation (DPI), respectively.Gross and microscopic lesions of small intestine, cecum and colon wereexamined and immunohistochemistry (IHC) staining was performed.Microscopic lesion severity was categorized as: 0=no lesion, 1=minimumvillous atrophy, 2=mild villous atrophy, 3=moderate villous atrophy and4=severe villous atrophy. IHC staining was scored as: 0=no signal,1=minimal staining, 2=mild staining, 3=moderate staining and 4=severestaining. Whole genome sequences of the viruses in original tissuehomogenate as well as at the passages P3, P7, P9, P25, P50, P65, P75 andP100 were determined using next-generation sequencing technology [13].

TABLE 6 Animal study design. Experimental infection of 5-day old pigletswith PEDV USA/IN19338/2013 at different cell culture passages. NecropsyNecropsy Group Virus Passage Inoculum (3 DPI) (7 DPI) G1 (n = 10) PEDVP7 10⁵ TCID₅₀/pig n = 5 n = 5 G2 (n = 10) PEDV P25 10⁵ TCID₅₀/pig n = 5n = 5 G3 (n = 10) PEDV P50 10⁵ TCID₅₀/pig n = 5 n = 5 G4 (n = 10) PEDVP75 10⁵ TCID₅₀/pig n = 5 n = 5 G5 (n = 10) PEDV P100 10⁵ TCID₅₀/pig n =5 n = 5 G6 (n = 10) Virus-negative Culture medium n = 5 n = 5 culturemedium DPI: Days Post Inoculation.

RESULTS AND DISCUSSION

Over serial passaging, viruses became more adapted to cell culture. Thehigher passages of PEDV USA/IN19338/2013 at P50, P75 and P100 grew moreefficiently and achieved higher infectious titers (FIG. 17).

The negative control piglets remained negative throughout the studyperiod. Piglets inoculated with P7, P25, P50, P75, or P100 virus all hadwatery diarrhea at 1-4 DPI and mild to moderate diarrhea at 5-7 DPI.Piglets inoculated with P7, P25, P50, P75, or P100 virus all shed virusin rectal swabs with a trend that piglets inoculated with higher passagevirus shed lower amount of viruses (FIG. 18). Specifically, virusshedding levels were: P7>P25>P50, P75, P100>Neg Ctrl (> meanssignificantly higher; but no significant differences among P50, P75 andP100 groups). There was a trend that piglets inoculated with lowerpassage virus had more severe villous atrophy. As shown in FIG. 19, at 3DPI, severity of microscopic lesions was P7, P25>P50, P75, P100, NegCtrl in duodenum; P7, P25, P50>P75, P100>Neg Ctrl in jejunum; and P7,P25, P50>P75, P100, Neg Ctrl in ileum. The villus-height-to-crypt-depthratios were: P7, P25, P50<P75, P100<Neg Ctrl in jejunum and P7, P25,P50<P75, P100, Neg Ctrl in ileum at 3 DPI (FIG. 20). At 3 DPI, the IHCstaining in jejunum was: P7, P25, P50, P75>P100>Neg Ctrl; the IHCstaining in ileum was: P7, P25, P50>P75, P100, Neg Ctrl; there were nosignificant differences of IHC scores between various viruses in cecumor colon (FIG. 21). Differences between groups at 7 DPI were not veryapparent.

Whole genome sequences were not only determined for viruses at thepassages P7, P25, P50, P75, P100 but also determined for the viruspresent in the original tissue homogenate as well as at the passages P3,P9 and P65. As shown in FIG. 16, comparisons of the whole genomesequences revealed that nucleotide and deduced amino acid changes duringserial passages up to P100 are mainly located in replicasenon-structural protein (nsp) 2, nsp3, nsp4, nsp5, nsp6, nsp15, spike(S), ORF3, envelope (E), membrane (M), and nucleocaspid (N) proteins. Itis notable that starting from the P25, a point mutation at 24908A>Toccurred in ORF3 which resulted in a truncated ORF3 protein due to earlystop codon of deduced amino acid translation; this point mutation wascarried over to the virus at P100. Taken into consideration of bothsequence changes and virus virulence changes during serial passages,amino acid changes at 15 positions may potentially be associated withvirus attenuation during serial passages in cell culture. These 15positions include: ppla protein 1564Ser>Phe, 1896Thr>Ile, 2600Asn>Tyr,3247Leu>Phe, and 3473Ala>Val; S protein 326Thr>Ile, 491Asn>Tyr,888Gly>Arg, 1277Leu>Phe, 1399Ile>Thr, and 1358Cys>Leu; truncated ORF3translation; E protein 69Leu>Ile; M protein 208Ala>Thr; and N protein439Thr>Ile.

In summary, the virulent U.S. PEDV prototype isolate USA/IN19338/2013clearly became less pathogenic during serial passages in cell culture.Clinical observations, virus shedding in rectal swabs, histopathologiclesions, and IHC staining data suggest that the P75 and P100 viruseshave been more attenuated than the P7, P25 and P50 viruses. However, theP75 and P100 viruses still can cause diarrhea and may not have beenattenuated enough to be an MLV vaccine candidate. Continuous passage ofthe virus in cell culture is needed to obtain a fully attenuated vaccinecandidate virus. Sixteen amino acid changes appear to be associated withvirus attenuation under the conditions of this study. This studyprovides a strong basis for developing a MLV PEDV vaccine and forunderstanding the molecular mechanism of virus attenuation.

Those skilled in the art will immediately recognize that althoughvariation exists in nucleotide and amino acid sequences among differentisolates, and that deletions and insertions may appear, nonetheless,sequence alignment techniques are well known so that any amino acidposition in a PEDV protein can be mapped to the appropriate amino acidposition in other isolates. Preferred algorithms and alignment programsfor this purpose include ClustalX version 2.0 (Larkin et al,Bioinformatics 23, 2947-2948 (2007)), BioEdit version 7.0.4.1 (Hall,Nucl Acids Symp Ser 41, 95-98 (1999)), and Lasergene software suite(DNASTAR Inc, Madison Wis.), which includes the Clustal W algorithm formultiple sequence alignment to align sequences and compare sequencesimilarity and differences.

FIG. 16 shows that the following amino acid changes are among thosepreferred for generating properly attenuated PEDV isolates, whichprovide for vaccine efficacy while exhibiting substantial safety: fromORF1 a and b, amino acid positions 814 (Val), 1076 (Val), 1564 (Phe),1896 (Ilu), 2310 (His), 2600 (Tyr), 3247 (Phe), 3473 (Val), 3522 (Arg);from the Spike gene, amino acid positions 257 (Asn), 326 (Ile), 375(Phe), 491 (Tyr), 881 (Arg), 888 (Arg), 1277 (Phe), 1339 (Thr), 1358(Leu); from ORF3, corresponding to amino acid position 39 or thereafter,any nucleotide change that provided a stop codon; from genome region E,69 (Ile) for the encoded envelope protein; for genome region M, 208(Thr) for the encoded membrane protein; and for genome region N, 141(Leu), 418 (Glu), 424 (Asp) and 439 (Ile) for the encoded nucleocapsidprotein. In addition to these changes, which can be used alone or incombination, those skilled in the art will recognize that equivalentamino acid changes are generally available, for example that positivelycharged residues can be substituted for positively charged residues(Arg, Lys, His), that negatively charged residues can be substituted fornegatively charged residues (Glu, Asp), or that equivalent substitutionscan be made based on polarity or functional group (such as Thr for Serand vice versa; Try for Trp and vice versa; Ileu, Val, Leu, all for eachother, all and the like).

REFRENCES

-   1. Paarlberg, P. L. 2014. Updated estimated economic welfare impacts    of porcine epidemic diarrhea virus (PEDV).    http://ageconsearch.umn.edu/bitstream/174517/2/14-4. Updated    %20Estimated %20Economic %20Welfare %20Impacts %20of %20PEDV.pdf.-   2. Grasland, B., Bigault, L., Bernard, C., Quenault, H., Toulouse,    O., Fablet, C., Rose, N., Touzain, F. and Blanchard, Y., 2015.    Complete genome sequence of a porcine epidemic diarrhea s gene indel    strain isolated in france in december 2014. Genome Announc 3.-   3. Mesquita, J. R., Hakze-van der Honing, R., Almeida, A., Lourenco,    M., van der Poel, W. H. and Nascimento, M. S., 2015. Outbreak of    Porcine Epidemic Diarrhea Virus in Portugal, 2015. Transbound Emerg    Dis 62, 586-8-   4. Pasick, J., Berhane, Y., Ojkic, D., Maxie, G., Embury-Hyatt, C.,    Swekla, K., Handel, K., Fairles, J. and Alexandersen, S., 2014.    Investigation into the role of potentially contaminated feed as a    source of the first-detected outbreaks of porcine epidemic diarrhea    in Canada. Transbound Emerg Dis 61, 397-410.-   5. Puranaveja, S., Poolperm, P., Lertwatcharasarakul, P.,    Kesdaengsakonwut, S., Boonsoongnern, A., Urairong, K., Kitikoon, P.,    Choojai, P., Kedkovid, R., Teankum, K. and Thanawongnuwech,    R., 2009. Chinese-like strain of porcine epidemic diarrhea virus,    Thailand. Emerg Infect Dis 15, 1112-5.-   6. Song, D. and Park, B., 2012. Porcine epidemic diarrhoea virus: a    comprehensive review of molecular epidemiology, diagnosis, and    vaccines. Virus Genes 44, 167-75.-   7. Vui, D. T., Thanh, T. L., Tung, N., Srijangwad, A., Tripipat, T.,    Chuanasa, T. and Nilubol, D., 2015. Complete genome characterization    of porcine epidemic diarrhea virus in Vietnam. Arch Virol 160,    1931-8.-   8. Stadler, J., Zoels, S., Fux, R., Hanke, D., Pohlmann, A., Blome,    S., Weissenbock, H., Weissenbacher-Lang, C., Ritzmann, M. and    Ladinig, A., 2015. Emergence of porcine epidemic diarrhea virus in    southern Germany. BMC Vet Res 11, 142.-   9. Theuns, S., Conceicao-Neto, N., Christiaens, I., Zeller, M.,    Desmarets, L. M., Roukaerts, I. D., Acar, D. D., Heylen, E.,    Matthijnssens, J. and Nauwynck, H. J., 2015. Complete genome    sequence of a porcine epidemic diarrhea virus from a novel outbreak    in belgium, january 2015. Genome Announc 3.-   10. Vlasova, A. N., Marthaler, D., Wang, Q., Culhane, M. R.,    Rossow, K. D., Rovira, A., Collins, J. and Saif, L. J., 2014.    Distinct Characteristics and Complex Evolution of PEDV Strains,    North America, May 2013-February 2014. Emerg Infect Dis 20,    1620-1628.-   11. Thomas, P. (2014). Field experiences using porcine epidemic    diarrhea virus (PEDV) vaccine in herds experiencing endemic disease.    In “2014 Iowa State University Swine Disease Conferencee for Swine    Practitioners”, pp. 38-42, Ames, Iowa.-   12. Schwartz, T. J. and Rademacher, C. J. (2015). Evaluation of the    effects of PEDv vaccine on PEDv naïve and previously PEDv-exposed    sows in a challenge model comparing immune response and preweaning    mortality. In “2015 ISU James D. McKean Swine Disease Conferencee”,    pp. 36-40, Ames, Iowa.-   13. Chen, Q., Li, G., Stasko, J., Thomas, J. T., Stensland, W. R.,    Pillatzki, A. E., Gauger, P. C., Schwartz, K. J., Madson, D.,    Yoon, K. J., Stevenson, G. W., Burrough, E. R., Harmon, K. M.,    Main, R. G. and Zhang, J., 2014. Isolation and characterization of    porcine epidemic diarrhea viruses associated with the 2013 disease    outbreak among swine in the United States. J Clin Microbiol 52,    234-43.-   14. Thomas, J. T., Chen, Q., Gauger, P. C., Gimenez-Lirola, L. G.,    Sinha, A., Harmon, K. M., Madson, D. M., Burrough, E. R.,    Magstadt, D. R., Salzbrenner, H. M., Welch, M. W., Yoon, K. J.,    Zimmerman, J. J. and Zhang, J., 2015. Effect of Porcine Epidemic    Diarrhea Virus Infectious Doses on Infection Outcomes in Naive    Conventional Neonatal and Weaned Pigs. PLoS One 10, e0139266.    Attenuation of U.S. S-INDEL-Variant Strain USA/IL20697/2014    (2014020697)

FIG. 24 and Tables 1 and 2, provide certain information concerning aminoacid changes that correspond to attenuates of a variant strainUSA/IL20697/2014. The variant isolate USA/IL20697/2014 was seriallypassed in cell culture in two independent lineages.

In the first lineage, the virus was passed in cell culture up to 60^(th)passage (P60); the viruses at different passages were named as2014020697-P1, 2014020697-P2, 2014020697-P3 . . . 2014020697-P60. Amongthem, whole genome sequences of the viruses 2014020697-P3 (SEQ IDNO:63), 2014020697-P5 (SEQ ID NO:8), 2014020697-P7 (SEQ ID NO:62),2014020697-P18 (SEQ ID NO:64), 2014020697-P30 (SEQ ID NO:65),2014020697-P45 (SEQ ID NO:39), and 2014020697-P60 (SEQ ID NO:66) weredetermined and compared.

In the second lineage, the virus was serially passed in cell culture andthe viruses at some passages were also plaque (colony) purified. Amongthem, whole genome sequences were determined and compared for theviruses 2014020697-P3R1 (SEQ ID NO:67), 2014020697-P5R1 (SEQ ID NO:68),2014020697-P7R1 (SEQ ID NO:15), 2014020697-P8R1 (SEQ ID NO:35),2014020697-P18R1 clone 89G8b (SEQ ID NO:36), 2014020697-P18R1 clone94F6a (SEQ ID NO:37), and 2014020697-P18R1 clone 92F6a (SEQ ID NO:37)(it should be noted that P18R1 94F6a and P18R1 92F6a (SEQ ID NO:37) areduplicates of the same clone run for verification purposes, see Tables 1and 2). Additionally, passaging of 2014020697-P18R1 G8b and2014020697-P18R1 F6a into subsequent passages 19 and 20 retained geneticidentity to their respective clones at passage 18R1. The resultantviruses provide both needed clinical safety, according to all recognizedtrial endpoints, and remain both highly protecting as to challenge bysimilar variant (INDEL) strains, and cross protective as to highlypathologic prototype strains.

Referring to the “Amino Acid Changes” section of FIG. 24 and Table 2, itcan be seen that ORF1a/1b at encoded amino acid position 551 providesleucine. In fact, leucine appears to be almost invariant in PEDVisolates (whether prototype or variant-INDEL) at this position, in thatleucine was found to represent residue 551 in approximately 500 randomlyselected compared published sequences. Although clinical data for2014020697-P18R1 F6a (SEQ ID NO:37), and 2014020697-P18R1 G8b (SEQ IDNO:36), both show remarkable levels of safety and efficacy, it is notedthat the 2014020697-P18R1 F6a does provide a further enhancement in bothsafety and efficacy, making it a highly commercializable material.2014020697-P18R1 F6a provides proline at position 551, an amino acidwell known to disrupt or define boundaries between peptide secondarystructural domain types, and thus the contribution of Pro 551 to thefinal phenotype would be expected. Given the invariant use of Leu atthis position in PEDV genomes, it is a further embodiment of the presentinvention to insert a Pro residue at position 551, or immediatelyadjacent thereto (such was within about 2-3 amino acid residues thereof,upstream or downstream. Modeling using well known algorithms (see P. Y.Chou et al., “Prediction of Protein Conformation”, Biochemistry, 13(2),pp 222-245, 1974; J. Gamier et al., J. Mol. Biol., v 120. pp. 97-120,1978; and J. Gamier et al., Methods Enzymology, v 266, pp. 540-543,1996) predicts an alpha helical domain at least involving residues DEDATimmediately upstream from the 551 position L, wherein L falls at (butstill contributes to) the approximate C-terminal end of this secondarydomain structural feature. Insertion of proline substantially disruptsthis alpha helical feature. It is also within the practice of theinvention to insert a glycine residue as amino acid 551 for ORF1a/b,again with the expectation of a resultant highly attenuated yet safevaccine, and the glycine residue may be similarly located within about2-3 amino acid residues of position 551, upstream or downstream. Allsuch substitutions are applicable to all PEDV clones, whether prototypeor variant (INDEL).

Turning now to the amino acid changes reflected in both 2014020697-P18R1G8b and 2014020697-P18R1 F6a at position 973 in the spike protein, itcan be seen that the wild type amino acid tyrosine has been replaced byhistidine, which substantially contributes to the valuable phenotype ofthese clones. It is therefore an embodiment of the invention to providehistidine at this locus generally, in all genomes of PEDV, which arebeing modified or selected for to provide safe and efficacious vaccines,whether from prototype or variant (INDEL) strains.

Turning further to an important amino acid change reflected in both2014020697-P18R1 G8b and 2014020697-P18R1 F6a at position 1009 in thespike protein (i.e. immediately after NIT in SEQ ID NO:47), it can beseen that the wild type amino acid serine has been replaced by proline.This mutation appeared during an earlier attenuating passage, between2014020697-P5R1 (SEQ ID NO:68) and 2014020697-P7R1 (SEQ ID NO:15). As issimilarly the case for the leucine to proline mutation noted above inregard of ORF1a/1b position 551, the appearance of proline is highlydisruptive of secondary and tertiary protein structure, andsubstantially contributes to the attenuated properties of the presentvaccine strains. It is again within the practice of the invention toprovide a proline residue at position 1009, or the correspondingposition in the spike protein of any PEDV vaccine virus. Glycine mayalso be substituted for proline at this position in any PEDV strain, inorder to improve vaccine safety. It is therefore an embodiment of theinvention to provide proline or glycine at this locus generally, in allgenomes of PEDV, which are being modified or selected for to providesafe and efficacious vaccines, whether from prototype or variant (INDEL)strains. It should also be noted that the proline/glycine replacementcan also be made immediately adjacent to position 1009, such as withinabout 2-3 amino acid residues thereof, upstream or downstream.

An additional feature of many of the viruses of the invention that areuseful as vaccines (including the G8b and F6a Passage 18R1 clones, SEQID NOS:36 and 37, and the separate lineage virus represented by Passage38, SEQ ID NO: 78, for example), is the expression therein of only atruncated ORF3 protein, typically caused by a frameshift mutation in theORF3 reading frame (see Table 2) and therefore the appearance of a stopcodon, or alternatively, the appearance of other features (such as adeleted amino acid, again see Table 2) resulting in a partially orwholly inoperative ORF3 protein. We have observed that deletions and/orframeshifts in ORF3 protein appear to be associated with adaptation totissue culture conditions, and therefore it is likely that ORF3 isnecessary for overcoming host immune defense, becoming unnecessary underin vitro culture conditions. As such, ORF3 deletions are important forvaccine viruses, contributing to their safety for use in swine.Referring to the truncated ORF3 protein expressed from 2014020697-P18R1G8b and 2014020697-P18R1 F6a (SEQ ID NO:31), it is noted (Table 2) thatthis mutation has already been successfully achieved by passage P8R1(see SEQ ID NO:18), with the resultant protein sequence being:

MFLGLFQYTIDTVVKDVSKSANLSLDAVQELELNVVPIRQASNVTGFLFTSVFIYFFALFKASSLRRNYIMLAARFAVIVLYCPLLYYCGAFLDATIICCTLIGRLCLVCFYSWRYKNALFIIFNTTTLSFLNGKAALTANPL

It is thus within the practice of the invention to provide vaccineviruses containing any truncation of the ORF3 reading frame, suitable tolimit ORF3 function, such as those already discussed above in relationto vaccine virus 2014020697-P38 (SEQ ID NO:78) and the earlier passagesthereof (see again Table 2), or those associated with virus sequencesSEQ ID NO: 36 and 37. Those skilled in the art are readily able toprovide a truncation of any ORF3 protein of any prototype strain orvariant INDEL strain, to containing corresponding truncations,referencing direct comparison of sequences and algorithms for aligningsequences. Thus, vaccine viruses of the invention include those havingSEQ ID NO:18 as resultant ORF3 protein, or any fragment thereof (10, 20,30, 40, 50, 60 amino acid residues, and the like), or any fragment offull length ORF3 protein that is less able to inhibit host immunefunction.

These mutations substantially contribute to the final clinicallyeffective phenotype of the vaccine attenuates of the present invention,and as aforementioned, referencing commonplace alignment programs andalgorithms, are readily copied into the amino acid sequence ofcorresponding proteins in all prototype and variant (INDEL) strains.Conservative amino acid replacements, instead of the specifically namedresidue changes, are also appropriate in all cases, and all amino acidchanges (and conservative substitutions thereof) observed for any of theprototype and variant (INDEL) viruses of the invention may be usefullyplaced in any PEDV virus (whether prototype or INDEL) for the purposesof improving the usefulness of any such resultant virus as a vaccine.

Further information on passaging herein is as follows. Subsequentpassaging (8 to 19) was performed from 2014020697-P7R1 (SEQ ID NO: 15)material. 2014020697-P8R1 consensus sequence did not show any changes atthe amino acid level compared to 2014020697-P7R1. 2014020697-P8R1 wasthen further passaged to 2014020697-P11R1, with clonal selection at eachstep between subsequent passages 11 through 14, followed by subsequentpassaging to 2014020697-P18R1 with collection of substantial material inorder to run clinical trials. The open reading frame amino acidsequences for 2014020697-P18R1 G8b and 2014020697-P18R1 F6a are shown asFIGS. 22B (SEQ ID NOS: 23-28) and 22C (SEQ ID NOS: 29-34), respectively,and the corresponding nucleotide sequences, full length, for2014020697-P18R1 G8b and 201402697-P18R1 F6a are shown as FIGS. 23B (SEQID NO: 36) and 23C (SEQ ID NO: 37) respectively. Significant andsuccessful clinical results are reported for 2014020697-P18R1 F6a. Inthis regard, see Examples 5 and 6, which shows the remarkable safety andcross protecting efficacy of these vaccine materials.

It should also be noted that “passage 7” 2014020697-P7, lineage 1 (SEQID NO:62) and “passage 60” 2014020697-P60 passage 60, lineage 1 (SEQ IDNO:39) amino acid sequence mutational information is also reported inFIG. 24 and Table 2, and mutations responsible for the safety andefficacy of this material are disclosed therein. That the material isalso an excellent candidate for a safe and efficacious cross-protectingvaccine is also apparent from the clinical data reported in Examples 5and 6, for 2014020697-P38 (SEQ ID NO:78), a precursor to 2014020697-P60and also the clinical data and discussion from Example 2 comparingmutations within 2014020697-P7 variant strain leading to attenuation.

Example 5 (Two Dose Study) Safety and Cross-Protection of PEDV INDELVirus Administered Orally at Approximately 1 and 21 Days of Age Followedby a Virulent PEDV Challenge

The objective of the study was to determine the safety and crossprotection of specific attenuate passages of PEDV isolates derived fromthe variant (INDEL) strain of PED, USA/IL/2014-20697, when administeredorally to piglets at 3 (+/−2) days of age (Day 0) and Day 21 followed bya virulent PEDv challenge at Day 35 (+/−2). Safety was determined by theincidence of mortality and clinical signs related to PEDv postinoculation at Day 0 and 21. Indication of cross-protection wasdetermined by the incidence of mortality and clinical signs related tovirulent PEDv post challenge. The tested viruses are those encoded fromDNA sequences: (a) SEQ ID NO:36 (designated Clone G6b, which remainsunchanged between passages 18 and 19 thereof); (b) SEQ ID NO:37(designated Clone F6, which remains unchanged between passages 18 and 19thereof); and (c) from a different set of passages from theUSA/IL/2014-2069 ancestor, Passage 38 (SEQ ID NO:78) thereof. It shouldbe noted that SEQ ID NOS 36 and 37 differ from each other only in thatencoded amino acid position 551 of polyprotein 1a/1b, is either L or Pat position 551 (see immediately after DAT in SEQ ID NO:46).

Vaccination doses (control was media only) of the live attenuates are1×104 TCID50 using 2 ML per oral doses. “TCID50” refers to “tissueculture infective dose” and is defined as that dilution of a virusrequired to infect 50% of a given batch of inoculated cell cultures.Various methods may be used to calculate TCID50, including theSpearman-Karber method which is utilized throughout this specification.For a description of the Spearman-Karber method, see B. W. Mahy & H. O.Kangro, Virology Methods Manual, p. 25-46 (1996).

The actual challenge material (having a concentration of 1×105 TCID50/5ML dose) is closely related to USA/Colorado/2013, GenBank Accession No.KF272920, being specifically a contemporary North American epidemicisolate from the University of Minnesota Veterinary DiagnosticLaboratory, Accession D13-031630, from a farm in Iowa, live material.The sample is negative for rotavirus A, B, and C, TGE, Clostridiumdifficile toxin and Clostridium perfringens.

PCR assessment of fecal shedding was performed by RTqPCR analysiswherein a reported value of 35 is negative (equal to a control) andvalues less than 35 are positive for shed virus. The number reported isthe number of cycles used for detection, with 35 being the maximum atwhich non-detection is declared. Protocol followed is generally asotherwise recited in the specification, and is similarly achieved asfollows. Fecal swabs are collected in 3 milliliters of DMEM media andstored at −80° C. until use. Samples are thawed at 4° C. and mixed byvortexing for 5 seconds. One milliliter of sample is removed from tubeand placed in a 96-well block and centrifuged at 3200 rpm for 10 minutesto sediment fecal particulate material. Two hundred microliters of fecalsample supernatant are used for nucleic acid extraction using the QiagenDSP Virus/Pathogen Mini Kit on the Qiagen QIAsymphony Automated Robot orthe Qiagen cador Pathogen 96 Kit on the QIA cube HT machine followingmanufactures instructions. Cycle threshold values were determined byRT-QPCR analysis using the Path-ID Multiplex One-Step RT-PCR kit(Applied Biosystems/Life Technologies) and PEDV N-gene primers andprobe. PEDV Forward Primer: PEDV N gene-F 5′-GAATTCCCAAGGGCGAAAAT-3′ @[100 uM], Reverse Primer: PEDV N gene-R 5′-TTTTCGACAAATTCCGCATCT-3′@[100 uM], PEDV Probe 6FAM 5′-CGTAGCAGCTTGCTTCGGACCCA 3′ TAMRA. Astandard curve is generated with the PEDV N-gene PCR amplicon and Ctvalues are used to determine a copy number value for each sample basedon the standard curve. Values reported in parenthesis show the number ofpiglets in each group that are positive for virus as numerator, with thedenominator showing the total number of piglets remaining in thatparticular group (not yet sacrificed for necropsy) on the daysindicated. Results are as follows:

It was noted (Table 7) that the Passage 38 material contributed to asmall amount of early mortality, not subsequently apparent, althoughsubsequent Passage 60 thereof (SEQ ID NO:66) was created to alleviatethis circumstance.

Referring to Table 8, fecal shedding was measured via PCR. Referencingthe “35” control value (no shedding), it can be seen that all clonesprotected the piglets compared to a challenged control that receivedonly media as vaccination. It should be noted that protection wasgreater for Clone F6a than for clone G8b, consistent with the completelyunique proline residue present in this clone (position 551 for Orf1a/bprotein).

Similarly, as shown in Table 9 (again compared to non vaccinatedcontrol), following challenge, all 3 viral isolates substantiallyprevented shedding shedding, with the F6a clone again showing betterresults than clone G8b.

TABLE 7 Cumulative Mortality by Time-point (animal deaths/percent oftotal) TX N D1 D4 D6 Total Media 30 0 0 0 0 (0.0) PED + 19 F6a 29 0 0 00 (0.0) PED + 19 G8b 28 0 0 0 0 (0.0) PED + 38 30 0 1 (3.3) 2 (6.7) 2(6.7)

TABLE 8 Fecal Shed in Piglets Post Challenge (RTqPCR (# pigspositive/total pigs)) TX D 36 D 37 D 39 D 42 D 45 D 47 D 49 D 52 D 55Media 35 35 (2/18) 27 (13/18) 25 (8/10) 29 (7/10) 32 (3/10) 31 (5/10) 31(5/10) 33 (3/10) PED + 19 F6a 35 35 35 35 35 35 35 35 35 PED + 19 G8b 3535 33 (2/17)  34 (2/8)  25 (4/8)  24 (7/8)  26 (6/8)  34 (2/8)  35(1/8)  PED + 38 35 35 35 35 35 35 35 35 35

TABLE 9 Duration of Shedding Post-Challenge (mean days positive (range))TX 1^(st) Inoculation 2^(nd) Inoculation Challenge Phase Media 0.1(0-2.0)   0.0 7.0 (0-17.0) PED + 19 F6a 12.9 (7.0-16.0) 4.4 (0-14.0) 0.0PED + 19 G8b 11.7 (0-16.0)   3.4 (0-14.0) 4.8 (0-17.0) PED + 38 11.5(2.0-16.0) 3.1 (0-13.5) 0.0

Example 6

Single Dose Efficacy and Safety in Day Old Piglets

The attenuated PEDV viruses corresponding to (encoded from) SEQ ID NO:36 (Clone G8b) and SEQ ID NO: 37 (clone F6a) show remarkable efficacy asvaccines, providing cross protection against later challenge withvirulent North American prototype virus (represented byUSA/Colorado/2013, GenBank Accession No. KF272920, for example). Suchprotection is achieved with only a single dose of vaccine, provided topiglets as early as at Day 1 of life, even when the mother sow isPEDV-naïve, i.e. seronegative, having been neither exposed to wildvirus, nor having ever been vaccinated. This level of efficacy accordsgreat flexibility to producers in terms of how swine operations aremanaged, when sows and piglets are vaccinated, and also accords toproducers the capacity to vaccinate sow herds or piglets withoutconducting assays to determine the sero-status of any animals prior tovaccination. These results are important given that the pathology ofPEDV infection is most serious in piglets at birth, and decreases (asdoes the need or value) of vaccination with age of the animals. Itshould be noted that the vaccines incorporating the SEQ ID NO:36 and 37viruses are useful irrespective of whether the mother sow isseropositive or seronegative prior to birth of the piglet, and thatcolostrum derived maternal antibody does not prevent the presentvaccines from being effective, irrespective of whether the mother sowwas previously infected with a wild strain, a vaccine strain (includingbeing infected thereby from the piglet), or was vaccinated with a liveor killed vaccine, in either case one or more times, includingpre-breeding and pre-farrowing. A second dose to the piglet can also begiven, if desired by the producer.

Accordingly, representative examples of vaccination programs useful inthe practice of the invention include: a method of treating orpreventing disease in a piglet caused by PEDV, comprising administeringto said piglet a first dose of the vaccine composition when said pigletis about 1-7 days old, further or optionally, administering a seconddose of said vaccine when the piglet is about 2-5 weeks old.Irrespective of whether 1 or 2 doses is administered to the piglet, theparent sow can be vaccinated pre-breeding or pre-farrowing in thealternative, or at both said time points, irrespective or whether themother sow was already seropositive (such was from previous infection).As aforementioned, alternatively, the mother sow is seronegative,

The objective of the present study was thus to determine the safety andcross protection of two passaged PEDV isolates (SEQ ID NOS: 36 and 37)derived from the variant (INDEL) strain of PED, USA/IL/2014-20697, whenadministered orally to piglets at 1 days of age (Day 0) followed by avirulent PEDv challenge at Day 21 (22 days of age). Safety will bedetermined by the incidence of mortality and clinical signs related toPEDv post inoculation. Indication of cross-protection was determined bythe incidence of mortality and clinical signs and intestinal lesionsrelated to virulent PEDv post challenge.

Vaccination doses (control was media only) are 1×10⁴ TCID⁵⁰ using 2 MLper oral doses. “TCID50” refers to “tissue culture infective dose” andis defined as that dilution of a virus required to infect 50% of a givenbatch of inoculated cell cultures. Various methods may be used tocalculate TCID50, including the Spearman-Karber method which is utilizedthroughout this specification. For a description of the Spearman-Karbermethod, see B. W. Mahy & H. O. Kangro, Virology Methods Manual, p. 25-46(1996).

Piglets within each group were further assigned to one of threeoutcomes, sacrificed for necropsy on Day 6, sacrificed for necropsy onDay 28, or the piglets complete the study and are reported out to Day40.

The actual challenge material (having a concentration of 1×10⁵ TCID₅₀/5ML dose) is closely related to USA/Colorado/2013, GenBank Accession No.KF272920, being specifically a contemporary North American epidemicisolate from the University of Minnesota Veterinary DiagnosticLaboratory, Accession D13-031630, from a farm in Iowa. The sample isnegative for rotavirus A, B, and C, TGE, Clostridium difficile toxin andClostridium perfringens.

Representative results are provided by fecal shedding scores, in theTable below. Dates below are calculated from Day 0 (i.e. 1 day of age,the date of vaccination with G8b or F6a attenuated virus).

TABLE 10 Fecal Shed in Piglets Post Virulent Challenge [RTqPCR (# pigspositive/total pigs)] Vaccine Group Day 25 Day 27 Day 30 Day 33 Day 37Day 40 T01 - 40  28 (14/19) 31 (7/9) 34 (6/9) 37 (4/9) 39 (1/9) Mediaonly T02 - 40 39 (1/18) 40 40 40 40 PEDV G8b T03 - 40 39 (1/21) 40 40 4040 PEDV F6a

PCR assessment of fecal shedding was performed by RTqPCR analysiswherein a reported value of 40 is negative (equal to a control) andvalues less than 40 are positive for shed virus. The number reported isthe number of cycles used for detection, with 40 being the maximum atwhich non-detection is declared. Protocol followed is generally asotherwise recited in the specification, and is similarly achieved asfollows. Fecal swabs are collected in 3 milliliters of DMEM media andstored at −80° C. until use. Samples are thawed at 4° C. and mixed byvortexing for 5 seconds. One milliliter of sample is removed from tubeand placed in a 96-well block and centrifuged at 3200 rpm for 10 minutesto sediment fecal particulate material. Two hundred microliters of fecalsample supernatant are used for nucleic acid extraction using the QiagenDSP Virus/Pathogen Mini Kit on the Qiagen QIAsymphony Automated Robot orthe Qiagen cador Pathogen 96 Kit on the QIA cube HT machine followingmanufactures instructions. Cycle threshold values were determined byRT-QPCR analysis using the Path-ID Multiplex One-Step RT-PCR kit(Applied Biosystems/Life Technologies) and PEDV N-gene primers andprobe. PEDV Forward Primer: PEDV N gene-F 5′-GAATTCCCAAGGGCGAAAAT-3′ @[100 uM], Reverse Primer: PEDV N gene-R 5′-TTTTCGACAAATTCCGCATCT-3′@[100 uM], PEDV Probe 6FAM 5′-CGTAGCAGCTTGCTTCGGACCCA 3′ TAMRA. Astandard curve is generated with the PEDV N-gene PCR amplicon and Ctvalues are used to determine a copy number value for each sample basedon the standard curve. Values reported in parenthesis then show thenumber of piglets in each group that are positive for virus asnumerator, with the denominator showing the total number of pigletsremaining in that particular group (not yet sacrificed for necropsy) onthe days indicated. Results at Day 27 and Day 30 show that a very highnumber of challenged (but not vaccinated) controls are shedding virus,whereas piglets vaccinated with attenuates G8b and F6a are remarkablywell protected.

Example 7 Introduction

Porcine epidemic diarrhea virus (PEDV) was detected in the United States(U.S.) for the first time in April 2013 [1]. Currently at least twogenetically different PEDV strains have been identified and co-circulatein U.S. swine (U.S. PEDV prototype strain and S-INDEL-variant strain)[2-4]. In a previous study, we have experimentally confirmed that theU.S. S-INDEL-variant strain is less pathogenic than the U.S. prototypestrain in 5-day-old piglets [5]. However, pathogenicity of PEDV can beage-dependent [6-7]. The objectives of the current study were to 1)evaluate the pathogenesis differences of two U.S. PEDV strains in weanedpigs; and 2) examine the cross-protection efficacy between two strainsin weaned pigs.

Materials and Methods

Eighty-five 3-week-old pigs were purchased from a conventional breedingfarm and delivered to the Iowa State University Laboratory AnimalResources facilities. All pigs were intramuscularly injected with a doseof Excede® upon arrival and confirmed negative for PEDV, porcinedeltacoronavirus, and transmissible gastroenteritis virus byvirus-specific PCRs on rectal swabs and negative for PEDV antibody by avirus-specific indirect fluorescent antibody (WA) assay on serumsamples.

Pigs were blocked by weight and then randomly divided into 7 groups with15 or 10 pigs per group (Table 11). Pigs were orogastrically inoculatedwith virus-negative culture medium (N), PEDV prototype isolateUSA/IN19338/2013-P7 (P-prototype strain), or PEDV S-INDEL-variantisolate USA/IL20697/2014-P7R1 (V-variant strain) at Day 0 (D0) followedby challenge at D28. Inoculation at each point used 10 ml of inoculumper pig, containing 104 TCID50/ml for virus inocula. Seven groups weredesignated according to 1st inoculation/2nd inoculation: P/V (15 pigs),V/V (15 pigs), N/V (15 pigs), P/P (10 pigs), V/P (10 pigs), N/P (10pigs), N/N (10 pigs).

TABLE 11 Experimental design. Day 0 (3-week-old) D4 D28 (7-week-old) D34D56 Group (1^(st) Inoculation) (Necropsy) (2^(nd) inoculation)(Necropsy) (Necropsy) G1 P/V (n = 15) Prototype PEDV n = 5 Variant PEDVn = 5 n = 5 G2 V/V (n = 15) Variant PEDV n = 5 Variant PEDV n = 5 n = 5G3 N/V (n = 15) Neg medium n = 5 Variant PEDV n = 5 n = 5 G4 P/P (n =10) Prototype PEDV n = 0 Prototype PEDV n = 5 n = 5 G5 V/P (n = 10)Variant PEDV n = 0 Prototype PEDV n = 5 n = 5 G6 N/P (n = 10) Neg mediumn = 0 Prototype PEDV n = 5 n = 5 G7 N/N (n = 10) Neg medium n = 0 Negmedium n = 5 n = 5 Notes: P = Prototype PEDV isolate USA/IN19338/2013; V= Variant PEDV isolate USA/IL20697/2014; N = Neg culture medium.Inocula: 10⁴ TCID₅₀/ml, 10 ml per pig for either the Prototype or theVariant isolates; or 10 ml culture medium per pig for Neg Control.

At D4, five pigs from the P/V, V/V, and N/V groups were necropsied forcomparison of gross and microscopic lesions between the two PEDVstrains. Five pigs from all 7 groups were necropsied 6 days after the2nd inoculation (D34) to evaluate cross-protection efficacy in term ofgross and microscopic lesions. The remaining 5 pigs per group were keptuntil 4 weeks after the 2nd inoculation (D56) to evaluate virus sheddingand post-challenge antibody response.

Clinical observations were recorded. Rectal swabs were collected at D0,2, 4, 7, 10, 14, 21, 28, 30, 32, 34, 38, 42, 49 and 56 and tested by aquantitative PEDV N gene-based real-time RT-PCR. Serum samples werecollected at D0, 7, 14, 21, 28, 35, 42, 49 and 56 and tested by indirectfluorescent antibody (IFA) assay and virus neutralization (VN) assayusing two PEDV strains as indicator viruses, respectively. Smallintestines were collected for histopathology evaluation andimmunohistochemistry (IHC) staining.

Generalized linear mixed (GLIMMIX) model was used to analyze virusshedding titer Log 10(gc/ml), IFA and VN Ab titers, clinical observationscores, pathological scores, villus heights to crypt depths (VH/CD)ratio differences among groups using Statistical Analysis System (SAS)version 9.3 (SAS institute, Cary, N.C.). During D0-D28, the N/N, N/V andN/P groups received the same inoculum and were analyzed as the sametreatment, similarly for the V/V and V/P groups, and P/V and P/P groups.IFA titers were transferred to log 2([IFA titer]/10), and VN titers weretransferred to log 2(VN titer) prior to statistical analysis. P-value<0.05 was defined as statistically significant difference.

Results

Pathogenesis comparison of U.S. PEDV prototype strain andS-INDEL-variant strain in 3-week-old pigs. During D0-D28, 7 groups ofpigs were in 3 categories: P-strain inoculation (P/V and P/P groups),V-strain inoculation (VN and V/P groups), and Neg culture mediuminoculation (N/V, N/P, and N/N groups).

Following the 1st inoculation (D0) of 3-week-old pigs, the two groupsinoculated with the P-strain (P/V and P/P) developed semi-watery towatery diarrhea between D2 and D4; one group (V/P) inoculated with theV-strain had mild soft feces during D2-D4 and the other group (V/V)inoculated with the V-strain developed watery diarrhea from D5 to D7.For the other 3 groups (N/N, N/V and N/P) inoculated with virus-negativeculture medium at the 1st inoculation, no diarrhea was observed up toD28.

As shown in FIG. 28, the Neg control pigs (N/N, N/V and N/P groups) didnot shed any virus in rectal swabs during D0-D28 as tested byquantitative PEDV rRT-PCR. Fecal shedding in the P-strain-inoculatedgroups (P/V and P/P) reached the peak level at D4 and then graduallydeclined. In contrast, fecal shedding in the V-strain-inoculated groups(V/V and V/P) gradually increased in the first a few days and reachedthe peak level at D7. Pigs inoculated with the P-strain (P/V and P/Pgroups) overall shed significantly higher amount of virus in feces thanpigs inoculated with the V-strain (V/V and V/P groups) from D0-D28(P=0.0396).

Five pigs from the P-strain-inoculation (P/V group), V-straininoculation (V/V group), and Neg medium inoculation (N/V group) werenecropsied at D4 (4 days post the 1st inoculation) to compare the macro-and micro-pathological lesions caused by the P-strain and V-strain in3-week-old pigs. The average content scores of small intestines, ceca,and colons were numerically higher in P-strain-inoculated pigs thanV-strain and mock-inoculated pigs but differences were not significant(FIG. 29A). The average gross lesion scores in small intestines and cecaof the V-strain and mock-inoculated pigs were not significantlydifferent, but were both significantly less severe than theP-strain-inoculated pigs (FIG. 29A). The villus-height-to-crypt-depth(VH/CD) ratios in distal jejunums and ileums of P-strain-inoculated pigswere significantly lower than V-strain pigs, suggesting that the villousatrophy caused by the P-strain was more severe than the V-strain (FIG.30A). Between V-strain groups and mock-inoculated groups, the averageVH/CD ratios in distal jejunums and ileums were not significantlydifferent (FIG. 30A).

For IHC staining at D4, P-strain-inoculated pigs had respective averageIHC scores of 4 and 4 in distal jejunum and ileum, which weresignificantly higher than the V-strain-inoculated pigs that had averageIHC scores of 1.4 and 1.4 in distal jejunum and ileum, respectively(FIG. 29B). Interestingly, PEDV IHC staining was also observed in cecumepithelia cells of 3 pigs (scores of 1, 1, and 2) and colon epitheliacells of 1 pig (score of 1) inoculated with the P-strain. PEDV IHCstaining was not observed in ceca and colons of V-strain-inoculatedpigs. PEDV IHC staining was not observed in any tissues (smallintestine, cecum, and colon) of mock-inoculated pigs.

Pathogenesis comparison of U.S. PEDV prototype strain andS-INDEL-variant strain in 7-week-old pigs. After the 2nd inoculation atD28 (pigs were 7-week-old at that time), the N/V, N/P and N/N groupswere compared to evaluate the pathogenesis of the P-strain and V-strainin 7-week-old weaned pigs.

After the 2nd inoculation, the N/P and N/N group did not have diarrheawhile the N/V group developed watery diarrhea during D33-D38 (5-10 dayspost 2nd inoculation). No any virus was detected from the N/N group inrectal swabs from D28 to D56. The N/V group overall shed significantlyhigher level of virus in rectal swabs than the N/P group during D28-D56(P=0.0359) (FIG. 28).

Five pigs necropsied from the N/N, N/V and N/P groups at D34 (6 dayspost the 2nd inoculation) were compared to evaluate the macro- andmicro-pathological lesions caused by the P-strain and V-strain in7-week-old pigs. Overall the macro-pathological changes were minor inall three groups of pigs. The average content scores of smallintestines, ceca, and colons were similar between the P-strain andmock-inoculated pigs but were significantly lower than theV-strain-inoculated pigs (FIG. 29B). The average gross lesion scores insmall intestines were not significantly different among the P-strain,V-strain and mock-inoculated pigs (FIG. 29B); but the average grosslesion scores in ceca and colons of the V-strain-inoculated pigs weresignificantly higher than the P-strain and mock-inoculated pigs (FIG.29B). At D34, the VH/CD ratios in distal jejunums and ileums werenumerically lower in the N/V group than the N/N and N/P groups but thedifferences were not significant (FIG. 31A).

At D34, only distal jejunums, ileums, and ceca of the N/V group and cecaof the N/P group were positive for PEDV IHC staining. The N/V group hadsignificantly higher average IHC scores in distal jejunums and ileumsthan the N/P and N/N groups (FIG. 31B).

Evaluation of cross-protection efficacy. Following the 2nd inoculationat D28, the 7 groups (P/V, V/V, N/V, P/P, V/P, N/P and N/N) werecompared to evaluate the effect of previous exposure (the 1stinoculation at D0) on the outcomes of the subsequent challenge (the 2ndinoculation at D28).

After the 2nd inoculation (D28), watery diarrhea was observed only inV/V and N/V groups between D33 and D38, and diarrhea was not observed inany other groups.

Fecal virus shedding was compared among 7 groups after the 2ndinoculation at D28 (FIG. 1). For 3 groups challenged with the V-strain,the P/V and V/V groups overall shed similar amounts of virus duringD28-D56 (P=0.9422); but both P/V and V/V groups shed significantly loweramounts of virus than the N/V group (P<0.0001). For 3 groups challengedwith the P-strain, the V/P and P/P groups shed significantly lessamounts of virus than the N/P group during D28-D56 (P<0.0001), yet theV/P group, which shed sharply higher amount of virus on D34 than theother days, was shedding significantly more viruses than the P/P groupfrom D28 to D56 (P=0.0001).

At D34 (6 days post challenge), among 3 groups challenged with theV-strain (NN, V/V and P/V groups), some minor macro-pathological changeswere only observed in the N/V groups but were not apparent in the V/Vand P/V groups. The average content scores and organ lesions in smallintestines were not significantly different among N/V, V/V and P/Vgroups. But the average content scores in ceca and colons as well as theaverage lesion scores in colons were significantly lower in the V/V andP/V groups than the N/V group (FIG. 29B). The V/V and P/V groups hadnumerically higher average VH/CD ratios in distal jejunums and ileumsthan the N/V group; however, significant differences were only observedbetween the N/V and V/V groups in distal jejunum and ileum VH/CD ratiosas well as between the V/V and P/V groups in distal jejunum VH/CD ratios(FIG. 31A). The V/V and P/V groups both had significantly lower averageIHC scores in distal jejunums and ileums than the N/V group (FIG. 31B).

At D34, among 3 groups challenged with the P-strain (N/P, V/P and P/Pgroups), macro- and microscopic pathological changes were not evident inany groups and no significant differences were observed between anygroups regarding the average content scores and tissue lesions (FIG.29B), average VH/CD ratios (FIG. 31A), or average IHC scores (FIG. 31B).

Antibody Responses.

After the 1st inoculation, all pigs inoculated with the P-strain (P/Pand P/V groups) or V-strain (V/V and V/P group) developed IFA and VNantibodies starting from D7-D14 regardless of which virus strain wasused as the indicator virus (FIG. 32A, 32B, 33A, 33B). Average antibodytiters peaked at D21 and D28 of all PEDV inoculated groups. PEDVantibody titers in these four groups increased slightly after thechallenge at D28 and then were maintained throughout the end of thestudy (D56). The N/P and N/V groups were PEDV antibody negative untilD42 (14 days post the 2nd inoculation) when IFA and VN antibodies becamedetectable and were maintained through D56. The N/N group remained PEDVantibody negative from D0-56.

SUMMARY

It appears that U.S. PEDV prototype strain achieved higher levels offecal virus shedding than S-INDEL-variant strain in 3-week-old pigs butthe opposite was observed in 7-week-old pigs. However, this observationneeds to be further confirmed to determine optimal ages at whichPrototype and INDEL vaccines provide peak efficacy in older pigs. U.S.PEDV prototype strain provided protection against challenge with bothhomologous and heterologous strains in weaned pigs; U.S. PEDVS-INDEL-variant strain provided protection against homologous strainchallenge and at least partial protection against heterologous(prototype) strain challenge in weaned pigs.

REFERENCES

-   1. Stevenson G W, Hoang H, Schwartz K J, Burrough E B, Sun D, Madson    D, Cooper V L, Pillatzki A, Gauger P, Schmitt B J, Koster L G,    Killian M L, and Yoon K J. (2013). Emergence of porcine epidemic    diarrhea virus in the United States: clinical signs, lesions, and    viral genomic sequences. Journal of Veterinary Diagnostic    Investigation 25(5): 649-654.-   2. Chen Q, Li G, Stasko J, Thomas J T, Stensland W R, Pillatzki A E,    Gauger P C, Schwartz K J, Madson D, Yoon K J, Stevenson G W,    Burrough E R, Harmon K M, Main R G, and Zhang J. (2014). Isolation    and characterization of porcine epidemic diarrhea viruses associated    with the 2013 disease outbreak among swine in the United States.    Journal of Clinical Microbiology 52(1), 234-243.-   3. Wang L, Byrum B, and Zhang Y. (2014). New variant of porcine    epidemic diarrhea virus, United States, 2014. Emerging Infectious    Diseases 20(5), 917-919.-   4. Vlasova A, Marthaler D, Wang Q, Culhane M R, Rossow K D, Rovira    A, Collins J & Saif L J. (2014). Distinct characteristics and    complex evolution of PEDV strains, North America, May    2013-February 2014. Emerging Infectious Diseases 20(10), 1620-1628.-   5. Chen Q, Gauger P C, Stafne M R, Thomas J T, Madson D M, Huang H,    Zheng Y, Li G, and Zhang J. Pathogenesis comparison between the    United States porcine epidemic diarrhea virus prototype and    S-INDEL-variant strains in conventional neonatal piglets. Journal of    General Virology (in press)-   6. Thomas J T, Chen Q, Gauger P C, Gimenez-Lirola L G, Sinha A,    Harmon K M, Madson D M, Burrough E R, Magstadt D R, Salzbrenner H,    Welch M W, Yoon K J, Zimmerman J J, and Zhang J. (2015). Effect of    porcine epidemic diarrhea virus infectious doses on infection    outcomes in naïve neonatal and weaned pigs. PLoS ONE    10(10):e0139266.-   7. Jung K, Annamalai T, Lu Z & Saif L J. (2015). Comparative    pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain    PC21A in conventional 9-day-old nursing piglets vs. 26-day-old    weaned pigs. Veterinary Microbiology 178, 31-40.

TABLE OF SEQUENCES SEQ ID NO: 1 S1 sequence of PEDV variant strain SEQID NO: 2 Spike gene 2014020697-P5 SEQ ID NO: 3 Spike gene 2013019338-P3SEQ ID NO: 4 Spike gene 2013022038-P3 SEQ ID NO: 5 Spike gene2013035140-P3 SEQ ID NO: 6 Spike gene 2013049379-P3 SEQ ID NO: 7 Spikegene 2013049469-P1 SEQ ID NO: 8 Whole genome 2014020697-P5 SEQ ID NO: 9Whole genome 2013019338-P3 SEQ ID NO: 10 Whole genome 2013022038-P3 SEQID NO: 11 Whole genome 2013035140-P3 SEQ ID NO: 12 Whole genome2013049379-P3 SEQ ID NO: 13 Whole genome 2013049469-P1 SEQ ID NO: 14Complete genome 2014020697-P5 SEQ ID NO: 15 Genomic sequence2014020697-P7R1 SEQ ID NO: 16 Genomic sequence 2014020697-P5 SEQ ID NO:17 2014020697-P8R1 ORF1a/1b polyprotein SEQ ID NO: 18 2014020697-P8R1spike protein SEQ ID NO: 19 2014020697-P8R1 ORF 3 protein (truncated)SEQ ID NO: 20 2014020697-P8R1 envelope protein SEQ ID NO: 212014020697-P8R1 membrane protein SEQ ID NO: 22 2014020697-P8R1nucleocapsid protein SEQ ID NO: 23 2014020697-P18R1 clone G8b ORF1a/1bpolyprotein SEQ ID NO: 24 2014020697-P18R1 clone G8b spike protein SEQID NO: 25 2014020697-P18R1 clone G8b ORF 3 protein (truncated) SEQ IDNO: 26 2014020697-P18R1 clone G8b envelope protein SEQ ID NO: 272014020697-P18R1 clone G8b membrane protein SEQ ID NO: 282014020697-P18R1 clone G8b nucleocapsid protein SEQ ID NO: 292014020697-P18R1 clone F6a ORF1a/1b polyprotein SEQ ID NO: 302014020697-P18R1 clone F6a spike protein SEQ ID NO: 31 2014020697-P18R1clone F6a ORF 3 (truncated) SEQ ID NO: 32 2014020697-P18R1clone F6aenvelope protein SEQ ID NO: 33 2014020697-P18R1 clone F6a membraneprotein SEQ ID NO: 34 2014020697-P18R1 clone F6a nucleocapsid proteinSEQ ID NO: 35 2014020697-P8R1 genomic sequence SEQ ID NO: 362014020697-P18R1 clone G8b genomic sequence SEQ ID NO: 372014020697-P18R1 clone F6a genomic sequence SEQ ID NO: 382014020697-P8R1 genomic sequence SEQ ID NO: 39 2014020697-P45 genomicsequence SEQ ID NO: 40 2014020697-P45 ORF1a/1b polyprotein sequence SEQID NO: 41 2014020697-P45 Spike protein SEQ ID NO: 42 2014020697-P45 ORF3protein SEQ ID NO: 43 2014020697-P45 Envelope protein SEQ ID NO: 442014020697-P45 Membrane protein SEQ ID NO: 45 2014020697-P45Nucleocapsid protein SEQ ID NO: 46 2014020697-P5 ORF1 polyprotein 1a/1bSEQ ID NO: 47 2014020697-P5 Spike protein SEQ ID NO: 48 2014020697-P5ORF3 protein SEQ ID NO: 49 2014020697-P5 Envelope protein SEQ ID NO: 502014020697-P5 Membrane protein SEQ ID NO: 51 2014020697-P5 Nucleocapsidprotein SEQ ID NO: 52 USA/IN19338/23013-P3 ORF1 polyprotein 1a/1b SEQ IDNO: 53 USA/IN19338/23013-P3 polyprotein 1a SEQ ID NO: 54USA/IN19338/23013-P3 Spike protein SEQ ID NO: 55 USA/IN19338/23013-P3ORF3 protein SEQ ID NO: 56 USA/IN19338/23013-P3 Envelope protein SEQ IDNO: 57 USA/IN19338/23013-P3 Membrane protein SEQ ID NO: 58USA/IN19338/23013-P3 Nucleocapsid protein SEQ ID NO: 59USA/IN19338/23013-P7 Genomic sequence SEQ ID NO: 60 USA/NC35140/2013-P7Genomic Sequence SEQ ID NO: 61 USA/NC49469/2013-P7 Genomic Sequence SEQID NO: 62 2014020697-P7 Genomic Sequence SEQ ID NO: 63 2014020697-P3Genomic sequence SEQ ID NO: 64 2014020697-P18 Genomic sequence SEQ IDNO: 65 2014020697-P30 Genomic sequence SEQ ID NO: 66 2014020697-P60Genomic sequence SEQ ID NO: 67 2014020697-P3R1 Genomic sequence SEQ IDNO: 68 2014020697-P5R1 Genomic sequence SEQ ID NO: 69 USA/IN19338/2013Homogenate Genomic sequence SEQ ID NO: 70 USA/IN19338/2013-P9 Genomicsequence SEQ ID NO: 71 USA/IN19338/2013-P25 Genomic sequence SEQ ID NO:72 USA/IN19338/2013-P50 Genomic sequence SEQ ID NO: 73USA/IN19338/2013-P65 Genomic sequence SEQ ID NO: 74 USA/IN19338/2013-P75Genomic sequence SEQ ID NO: 75 USA/IN19338/2013-P100 Genomic sequenceSEQ ID NO: 76 2014020697-P19R1 G8b Genomic sequence SEQ ID NO: 772014020697-P19R1 F6a Genomic sequence SEQ ID NO: 78 2014020697-P38Genomic sequence

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

The inventions being thus described, it will be obvious that the samemay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions.

We claim:
 1. An isolated Porcine Epidemic Diarrhea Virus (PEDV) encodedby a DNA polynucleotide sequence that is SEQ ID NO: 36 or SEQ ID NO: 37.2. A vaccine composition comprising a porcine epidemic diarrhea virus(PEDV) according to claim 1, and a carrier, wherein said composition iscapable of protecting swine from challenge by both variant and prototypestrains of PEDV and preventing or treating one or more symptomsassociated with PEDV infection, and wherein achievement of protection isdetermined by an endpoint selected from the group consisting ofprevention or control of any of the PEDV infection symptoms ofdehydration, fever, diarrhea, vomiting, poor lactational performance,poor reproduction performance, mortality, and prevention or control ofweight loss or failure to gain weight.
 3. The vaccine composition ofclaim 2 wherein the virus is live or killed.
 4. The vaccine compositionof claim 2 wherein said carrier is a diluent.
 5. The vaccine compositionof claim 2 further comprising an adjuvant.
 6. The vaccine composition ofclaim 2 wherein said protected swine include any of sows, gilts, boars,hogs, and piglets.
 7. The vaccine composition of claim 5 wherein theadjuvant is de-oiled lecithin dissolved in an oil and aluminumhydroxide.
 8. The vaccine composition of claim 5, wherein said adjuvantis CpG/DEAE-dextran/mineral oil (TXO).
 9. A method of preventing diseasein a piglet caused by PEDV, said method comprising administering to saidpiglet the vaccine composition of claim 2, wherein a first dose of saidvaccine is administered when the piglet is about 1-7 days old.
 10. Themethod of claim 9, wherein said administering further comprisesadministering a second dose of said vaccine when the piglet is about 2-5weeks old.
 11. The method of claim 9, wherein 2 doses are administeredto the piglet, and the parent sow, although vaccinated pre-breeding, wasnot vaccinated pre-farrowing.
 12. The method of claim 9, wherein 2 dosesare administered to the piglet, and the parent sow is vaccinatedpre-farrowing.
 13. The method of claim 9, wherein only a single dose isadministered to the piglet, and wherein the mother sow of the piglet isnaïve to PEDV, and is not, at any time, vaccinated.
 14. An isolatedPorcine Epidemic Diarrhea Virus (PEDV) that is encoded by a DNApolynucleotide sequence that is at least 95% or 99% identical, at a fulllength nucleotide level, to SEQ ID: NO:37, wherein said nucleotidesequence comprises one or both of (1) an ORF1a/1b protein having aproline residue at the amino acid position thereof corresponding toposition 551 in SEQ ID NO:46; and (2) a spike protein having a histidineresidue at the amino acid position thereof corresponding to position 973in SEQ ID NO:
 47. 15. A full length RNA polynucleotide that correspondsto the encoding DNA polynucleotide of claim 1, or the complementthereof.
 16. The RNA polynucleotide of claim 15 that is an infectiousclone.
 17. A plasmid or bacterial artificial chromosome that comprisesthe encoding DNA polynucleotide of claim 1.